DESIGN OF ENZYME INHIBITORS

Importance of enzyme inhibitors- Half of top 20 drugs in the world are enzyme
inhibitors.

Current design approaches- Knowledge of enzyme mechanisms & structures

used in design process.

Enzyme inhibitors in medicine- Inhibitors of enzymes are potential drugs as

they selectively bock certain metabolic pathways decreasing concentration of
enzymatic products or increasing concentration of enzymatic substrates.

Effectiveness of an enzyme inhibitor depends on its potency, selectivity and

specificity. Good specificity prevents depletion of inhibitor by non specific
pathways. Toxicity is minimized if interaction of other enzymes can be
minimized. Good bioavailability is also important. Highly polar or charged
compounds cannot readily cross cell membranes and are less useful as drugs
(liposomes useful here).
 Agents against against microbes may be such that-
1. They involve inhibition of enzyme essential to microbe
which is different or does not exist in the host.Eg.-
2) Enzymes-different in host & microbe- isozymes.
• Highly specific inhibitors exploit subtle structural
differences between these isozymes. Eg-
• Methotrexate-

O OH
O
OH
NH2 N
H
N O
N N

H2N N N

These drugs should kill tumour cells without harming normal cells .These
are difficult to design. Most drugs are anti proliferative with lots of side
effects to actively multiplying cells.
3) Drugs which treat enzyme dysfunction or imbalance of metabolites
 OTHER USES OF ENZYME INHIBITORS:-
1. Tools for elucidation of structure and function of enzymes.
2. Mimic genetic disease in animal models.
3. Screening of microbes for inhibitor resistant mutants.

CLASSIFICATION OF ENZYME INHIBITORS-

 Noncovalent inhibitors-
• Rapid reversible inhibitors.
• Tight, slow, slow-tight binding inhibitors.
• Transition-state analogs.
• Multisubstrate analogs.

 Covalent inhibitors-
• Mechanism based inhibitors.
• Affinity labels.
• Pseudoreversible inhibitors.
 RATIONAL DESIGN OF
INHIBITORS-

1.Traditional methods:- Inhibitors were analogs

of substrates, products or reaction
intermediates.
2.QSAR studies:- Better predictions as to which
changes would lead to better activity.
3.CADD:- 3D structure of enzyme or
pharmacophore structure has to be known.
 FORCES IN THE FORMATION OF ENZYME-INHIBITOR COMPLEXES-
• In order to design better inhibitors, basic knowledge of the binding forces between an enzyme
active site and its inhibitors are required.
• The reversible binding process of an inhibitor to an enzyme active site can be shown by-

Lower Ki values indicate high [E.I]. So better inhibition.

Alternative way of describing affinity of inhibitor for enzyme Gibb’s free

energy (∆G)
∆G= RT ln Kd
Where Kd= dissociation constant under consideration, here Ki
R= Universal gas constant, T= temperature in K
• The more negative the ∆G values, the smaller is the Ki
Hence, larger the attraction forces.
• ∆G can also be expressed in terms of enthalpy (∆H) and entropy (∆S)
∆G= ∆H- T∆S
Hence ∆G is decreased by decrease in enthalpy or increase in entropy.
• From a qualitative view, degree of the binding affinity and specificity depends on (is a sum
of) all existing
– Electrostatic forces
– Dispersion forces
– Hydrophobic interactions
– Hydrogen bonds
1) Electrostatic forces

• These describe interactions between dipolar or charged atoms and

molecules. Magnitude of these forces depends strongly on the dielectric
constant of the surrounding medium.

• Dielectric constant of water = 80, EtOH = 24, Benzene = 2.3

• High polarity of water causes “leveling effect” and greatly diminishes

attraction or repulsion between any two charged groups.

• The above reaction illustrates the presence of “salt bridges” between two
oppositely charged molecules. It shows that for I-E to complex,
desolvation of both ions is necessary at the cost of enthalpy, but leads to
a significantly favorable increase in entropy by the freed water molecule.

• As there are two opposite energy interactions, in such a complex system,

it is difficult to evaluate the contribution of of each individual energy.
2) Dispersion forces (Non polar or Vander waals interactions)

• Based on induced dipoles between nonpolar molecules. Magnitude of

these depends on the polarizability of atoms involved. Weak but additive
forces.
• E.g.: Not between O-H and H2C=CH2 but between two H2C=CH2
groups.
• However in non polar moieties, hydrophobic interactions play a major
role and dispersion forces, a very minor role.

3) Hydrophobic interactions
• Hydrophobic interactions leads to decrease in entropy as water
molecules surround the organic molecule with a high degree of order,
in order to optimize the hydrogen bonding.
• No large changes in enthalpy are observed.
4) Hydrogen bonds
•Significant in non polar solvents. However, water greatly diminishes their
magnitude.
•To form a hydrogen bond between E & S, E-H2O hydrogen bonds and S-
H2O bonds have to be broken.
•Hence, net energy gain is very small or zero. However, formation of E-I
adduct usually leads to overall increase in entropy.
•The contributions of all these forces to the overall binding affinity of the
inhibitor to the enzyme are very difficult to estimate quantitatively.
•Hydrophobic interactions, electrostatic forces and hydrogen bonding seem to
contribute to a favorable increase in entropy. Enthalpy estimations are much
more complex.
•If an inhibitor is buried i.e. sequestered from water in the active site, all these
forces generally become very potent.
 RAPID REVERSIBLE INHIBITORS:-
• Prevent substrate binding, by binding to enzymes’ active site in a rapid,
reversible fashion. Design of potent inhibitors of this class strives to
optimize non covalent binding forces eg. electrostatic forces, hydrogen
bonds, hydrophobic interactions, dispersion forces between inhibitor and
enzyme active site. So information about shape and chemical nature of
the active site and areas in close proximity is necessary.
• To improve potency (rational design starting from lead) :
1) Areas of bulk tolerance must be identified hydrophobic interactions
eg. Phenyl group in hydrophobic pocket etc.
2) H-bonding and salt bridges at strategic positions especially “buried”
interactions.
KINETICS:-
1. Kinetic evaluation of an inhibitor can provide information regarding
nature of enzyme – inhibitor eg.
2. Site of binding
3. Sequence of binding of inhibitor and substrate (s) in multisubstrate –
catalyzed reactions.
4. Effectiveness of binding (Ki) values.
 TYPES OF RAPID REVERSIBLE INHIBITORS

Competitive Inhibitors:
Compete for the same binding site therefore mutually exclusive binding.
Uncompetitive Inhibitors:
Do not bind to free enzyme.
Noncompetitive inhibitors: Bind with same affinity to the free
enzyme and enzyme- substrate complex.
Majority of drugs: Reversible enzyme inhibtor.
Scheme for enzyme-substrate-inhibitor equilibria

Ki – Inhibitor dissociation constant for EI complex.

Ks – Substrate dissociation constant for ES complex
αKi –inhibitor dissociation constant for ESI complex
 COMPETITIVE INHIBITORS:
• Usually common binding site.
• Allosteric mechanism also possible

•Michaelis- Menten equation becomes

Therefore as [I] increases apparent value of Km increases. The Vmax is not

affected, Vmax is reached when sufficient large excess of substrate is
present.
 UNCOMPETITVE INHIBITORS
• Uncompetitive inhibitor binds exclusively to the ES complex and have no
affinity for free enzyme
• Rare in unisubstrate catalysed reaction.
• In bisubstrate catalysed reaction, inhibitor might be competitive with
respect to one substrate and incompetitive with respect to the other
• Uncompetitve inhibitor decreases Vmax by a factor of (1+ [I]/ Ki ] because
some of the enzyme remains in E.S.I from even at infinite substrate conc.
• The apparent KM also decreases by the same factor i.e (1+ [I]/Ki)
• Because of exclusively bindng to the ES complex the IC50 of these
inhibitors decreases with increasing [S]
• IC50= αKi ( 1+ KM/[S])

OR
 NONCOMPETITIVE INHIBITORS
These bind to free enzyme as well as to enzyme substrate complex
Ki - Dissociation constant for E+I  E.I
αKi – Dissociation constant for ES+ I  E.S.I
In ideal cases α= 1 & Ki = αKi
The general vel. Equation

if α= 1, then the equation can be written as

When α ≠ 1, then the inhibitors are referred to as “mixed inhibitors”

The ideal inhibitor- KM unchanged, Vmax altered
Mixed inhibitor- Vmax & KM altered
Since non competitive inhibitor can bind to E.S complex, the IC50 of these is
not significantly influenced by substrate concentration.
 SLOW, TIGHT & SLOW-TIGHT BINDING INHIBITORS

Michaelis- Menton Kinetics not necessarily true here

Concentration of inhibitor not >>> concentration of enzyme ( unlike R.R.I )
Equilibria achieved slowly ( secs or minutes)
Dissociation rates of complex i.e. E.I E +I are slow while association rates of
complex i.e. E+ I E-I may be slow or fast i.e. slow equilibria rate not
necessarily slow binding rate.
Simple slow binding inhibitor- inhibitor concentration very high than enzyme
concentration for inhibition to occur.
Slow-tight binding- very strong affinity for enzyme- inhibitor concentration
comparable to enzyme concentration.
In this case inhibitor concentration is not independent of enzyme concentration
therefore Michaelis-Menten kinetics not applicable.
When dissociaition rates of complex that is E.I E+ I approaches zero then
inhibitor becomes functionally irreversible.
 SLOW-BINDING INHIBITORS
(Competitive inhibitors only discussed)
Two different mechanisms suggested to rationalize slow-binding behaviors:
1] Direct binding process of inhibitor to enzyme is slow
Reason: barrier at binding site.
On correct alignment binding is tight and release from enzyme is even more
slow.

2] Initial equilibria: E + I E.I is fast, but subsequent rearrangement to form stable

enzmye- inhibitor complex E.I* is slow
i.e. Ki*( K4/K-4) < Ki ( K3/K-3)
i.e. enzyme is better equipped to accommodate inhibitor in transition state as
compared to ground state.
Consequently- tight binding results. Therefore inhibitor released extremely
slowly from enzyme.
(Alternative explanation- release of H2O molecules slow…..not likely)
Advantage of these: E.I* E.I is independent of substrate concentration
therefore no reversal of inhibition occurs.
• Eg. Ramiprilat- slow-tight inhibitor of ACE
 TRANSITION STATE ANALOGS
Enzymes stabilize transition state structure by a variety of strategies:
Approximation: Bringing together the substrate molecules and reactive groups of enzyme
active site into correct orientation and proximity e.g

Covalent catalysis: Formation of a covalent bond between the enzyme reactive group and
substrate.
Usually either a) nucleophilic: electrons from amino acid of enzyme e.g. serine –OH,
cysteine, aspartate, lysine, histidine, tyrosine.
b) Metal ions or cofactors generally involved as aminoacids do not have electrophilic
groups. E.g. Zinc metaloenzymes e.g. matrix metalloproteases
(See pg 338, 340, 341 of R.A.Copeland et al for diagrams)
Conformational distortions: Steric and electronic changes in active site are required
Acid-base chemistry i.e. proton transfer
Inhibitors can be designed using similar strategies.
Transition state analogs:
These are stable compounds which mimic the structure of the activated complex
during the transition state of an enzymatic reaction.
Transition state analogs take advantage of the extra binding energies available in
transition state but not in the ground state.
Principle: Rate of enzyme catalysed inhibition is correlated with rate of a
noncatalysed inhibition by the same factor as the affinity of enzyme for the
transition state to affinity of enzyme for the substrate.

Therefore enzymatic catalysis (kE/kN) is proportional to the enhanced binding of

transition state to the enzyme (kT/ kS). kT/ kS can be of the order 108 to 1014
 CHALLENGES IN DESIGNIG A TRANSITION STATE ANALOG
1. Mechanism of target enzyme should be known
2. Energy profits of enzyme should be known
3. Analog for metastable structure- inherent difficulty

The analog must have fully developed binds in contrast to the transition
state, where partial bonds are possible. Therefore these compounds can
be more like the high energy intermediates of a normal reaction.

e.g. 1) Adenosine deaminase converting adenosine (I) into inosine (III)

has been popular target enzyme for design of transition state inhibitors.

The mechanism of cataysis is proposed to proceed via the hydrated

tetrahedral intermediate (II). The transition state is in turn proposed to
resemble this intermediate. Nebularin (IV) binds in the form of
Nebularin- 1,6- hydrate (V) to enzyme’s active site with Ki value ≈
dissociation constant of transition state of enzyme-adenosine complex.
Another example id 8R isomer of 2’- deoxycoformycin (VI).
• e.g. 2) 2- (Dimethylamino)- 1- ethyl diphosphate (i) was designed as
areaction intermediate/ transition state analog of Isopentenyl diphosphate
isomerase. The enzyme catalyzes the reversible conversion of isopentenyl
diphosphate (ii) to dimehthyl allyl diphosphate (iii). The reaction is
proposed to proceed via the carbocationic intermediate (iii)
 MULTI SUBSTRATE INHIBITORS
• Many enzymatic reactions require simultaneous binding of two or more substrates
at the active site in order for catalysis to occur. The bound substrates are required to
have particular orientations as well as be in close proximity to each other.
• The design of multi-substrate inhibitor mimics simultaneous binding of two or more
substrates at the active site of the enzyme.
• Thus, 2/3 substrates/ substrate analogs are covalently linked in these inhibitors, to
form bisubstrate/ trisubstrate analogs.
• The Ki value for a bisubstrate analog should be in the range of the product of the
KM or Ki values of the 2 substrates or substrate analogs respectively. Therefore the
ideal multisubstrate inhibitor should be able to bind 108 times as tightly as the
product of the substrate binding constants. However this has not been achieved in
practice.
• An ideal multisubstrate inhibitor is also expected to exhibit competitive inhibition
patterns with each of the subnstrates, as binding of inhibitor should be mutually
exculsive with both substrates.
THE REASON FOR ENHANCED BINDING IS AS FOLLOWS:
• Entropic advantage of reduced molecularity-
• Binding process of two single substrate analogs is accompanished by the loss of
two sets of transitional and overall rotational entropies, whereas in the
multisubstrate analog, only one set of transitional and rotational entropy is lost.
• However, binding enthalpies, electrostatic, H- bonds, dispersion forces as well as
entropies due to release of water molecules remain the same.
 Another concept related to multisubstrate inhibition-
• Use of a hydrophobic anchor at the binding site near that of the enzyme active
site e.gHMG-CoA

(a) Compactin (b)

A class of multi-substrate inhibitors involves use of hydrophobic anchors which takes
advantage of hydrophobic binding interactions near active site of enzymes e.g. HMG-CoA
reductase which catalyses the reaction below:
The binding behaviour is similar to that of multi-substrate inhibitor by simultaneously
binding to two binding sites on the enzyme leading to greatly enhanced inhibiton.
 IC50 VALUES
In order to screen a large no. of samples quickly, potencies of enzyme
inhibitiors are frequently compared using IC50 values rather than Ki
values.
The [S] and [E] are kept constant and [I] is varied in 10 fold increaments.
IC50 – [I] which leads to 50% enzyme inactivation.
Therefore IC50 values are not constant except in case of non competitive
inhibitor, but dependent on [S] used in the experiment.
Therefore for competitive inhibitor,
For uncompetitive inhibitor, Therefore when type of inhibition is unknown,
then [S] CLOSE TO KM should be used for estimation.
IC50 values have last used in case of mixed competitive and irreversible
inhibitor.
Therefore double reciprocal plot shows a series of
Formula
Lines that intersect at Y-axis
Formula
Examples: DHFR inhibitors: Methotrexate & Trimethoprim
• Lovastatin: transition state mimic-tight binding enzyme inhinitor
• ACE inhibitors: transition state mimic-tight binding enzyme inhinitor