This document discusses rational design of non-covalently binding enzyme inhibitors. It begins by classifying these inhibitors based on kinetics into rapid reversible inhibitors like competitive, uncompetitive and noncompetitive inhibitors. It also classifies them based on structure and mechanism into ground state analogs, multisubstrate analogs and transition state analogs. The key forces involved in enzyme-inhibitor complex formation are then explained like electrostatic interactions, hydrogen bonding, hydrophobic interactions and van der Waals forces. Details of steady state enzyme kinetics using Michaelis-Menten equation and different plots for kinetic data analysis are provided. Examples of different classes of reversible inhibitors are also given.

1. Rational Design of Non-covalently Binding Enzyme Inhibitors
Advanced Medicinal Chemistry (Unit 4)
- Prepared by
Chandni Pathak
M.Pharm (Pharmaceutical
Chemistry - 1st Sem)
Parul Institute of Pharmacy

2. Contents
Introduction
Classification of Non-covalently binding
enzyme inhibitors
A] On the basis of Kinetics
1. Forces Involved in forming the
Enzyme-Inhibitor Complex
1.1 Electrostatic Interactions
1.2 Van der Waals forces
1.3 Hydrophobic Interactions
1.4 Hydrogen Bonds
1.5 Cation - π bonding
1. Steady State Enzyme Kinetics
2.1 Michaelis Menten equation
2.2 Treatment of Kinetics Data
2
3. Rapid, Reversible Inhibitors
3.1 Competitive Inhibitors
3.2 Uncompetitive Inhibitors
3.3 Noncompetitive Inhibitors
B] On the basis of Structure/Mechanism
1. Ground-state Analogs
2. Multisubstrate Analogs
3. Transition state Analogs
References

3. Introduction
➢ Noncovalently binding enzyme inhibitors binds to the enzyme’s active site
without forming a covalent bond.
➢ The affinity and specificity of the inhibitor depends on the combination of
electrostatic and dispersive forces, and hydrophobic and hydrogen-bonding
interactions.
3

4. Classification of Non-covalentlybinding enzyme inhibitors
A] On the basis of kinetics:
1)Rapid reversible inhibitors
2)Tight-binding inhibitors
3)Slow-binding inhibitors
4)Slow-tight-binding inhibitors
5)Irreversible inhibitors
6)Pseudoirreversible inhibitors
B] On the basis of structure:
1) Ground-state analogs
2) Multisubstrate inhibitors
3) Transition state analogs
4

5. 1. Forces Involved in Forming the Enzyme-Inhibitor Complex
1.1 Electrostatic (Ionic) Interactions
1.2 Ion-dipole and Dipole-dipole Interactions
1.3 Hydrogen Bonding
1.4 Hydrophobic Interactions
1.5 Van der Waals Interactions
5

6. 1.1 Electrostatic Interactions
● It involves ion-ion, ion-dipole and dipole-dipole interactions.
● At physiological pH, the side chains of basic residues such as lysine and arginine, and to a
lesser extent, the imidazole ring of histidine, will be protonated, whereas the acidic groups
on the side chains of the aspartic and glutamic acid residues will be deprotonated.
● Also, the N-terminal amino groups and the C-terminal carboxylates will be ionized.
● Therefore, in addition to atoms with permanent and induced dipoles, an enzyme potentially
will have several charged groups available for binding to charged or polarized groups on a
substrate or inhibitor.
6

7. 1.1 Electrostatic Interaction (contd)....
● The electrostatic force (F) between the charged atoms (q1 and q2) will depend on the distance between the charged groups (r) and the
dielectric constant of the surrounding medium (D).
● The strength of an ion-ion interaction is inversely related to the square of the distance (r2) between the ions. Similarly, the strengths of ion-
dipole and dipole-dipole interactions have 1/r4 and 1/r6 respectively.
● As the strength of the interaction decreases more slowly with distance, ion-pair interactions can be thought of long range interactions.
● Conversely, interactions involving dipoles are effective over only a short range.
● Hence, the dependency of the strength of interaction on the distance between atoms is an important consideration when designing potential
enzyme inhibitors.
7

8. 1.2 Van der Waals forces/ Nonpolar interactions/ London Dispersion forces
● As two molecules closely approach each other, there is an interpenetration of their
electron clouds.
● Temporary local fluctuations in electron density occurs, giving rise to temporary
dipole moment, although the molecules may not have net dipole moment.
● Thus there will be an attractive force between the two molecules, with the
magnitude of the force depending on the polarizability of the particular atoms
involved and the distance between each other.
● These forces are more stronger between 2 nonpolar compounds than between
nonpolar compounds and water.
8

9. 1.2 Van der Waals forces/ Nonpolar interactions/ London Dispersion forces (contd)....
● The optimal distance between the atoms is the sum of each of their van der
Waals radii, so these forces come into play only when there is good
complementarity between enzyme and inhibitor.
● Although van der Waals forces are quite weak, usually around 0.5-1.0
kcal/mol for an individual atom-atom interaction, they are additive and can
make an important contribution to inhibitor binding.
9

10. 1.3 Hydrophobic Interactions
● When a nonpolar compound is dissolved in water, the strong water-water interactions around
the solute lead to an effective ‘ordering’ of the structure of the solvent. Due to this, there is
negative entropy of dissolution.
● When a nonpolar inhibitor binds to a nonpolar region of an enzyme, all the ordered water
molecules become less ordered as they associate with bulk solvent, leading to an increase in
entropy.
● Increase in entropy leads to decrease in free energy through stabilization of the enzyme-
inhibitor complex.
● It has been calculated that a single methylene-methylene interaction releases about 0.7
kcal/mol of free energy.
10

11. 1.4 Hydrogen Bonds
● A hydrogen bond occurs when a proton is shared between two electronegative atoms.
● The energy of the amide-amide NHO hydrogen bond is about 5 kcal/mol.
● For a hydrogen bond to form between an enzyme and an inhibitor, any hydrogen bonds between the inhibitor and water, as well as those between the enzyme and water,
must be broken.
● Overall, the total number of hydrogen bonds remains constant and, provided that the hydrogen bonds between the inhibitor and enzyme are not significantly more favorable
than those between water and the inhibitor or those between water and the enzyme, the net change in enthalpy is usually insignificant.
● On the other hand, formation of the enzyme-inhibitor complex usually leads to an overall increase of entropy because the inhibitor remains bound to the enzyme and the
formerly bound water molecules are released.
11

12. 1.5 Cation - π bonding
● Cations, from simple ions (Li+) to more complex organic molecules (ACh) are
strongly attracted to the electron rich (π) face of any aromatic compounds.
● These interactions are common in protein data bank and it has been
estimated that more than 25% of tryptophan residues are involved in
interactions of this type.
● The finding that the cationic group of acetylcholine was bound primarily by
aromatic residues, most especially by a tryptophan residue, not by the
expected carboxylate anion, provided evidence that cation-π interactions may
play an important role in ligand binding.
12

13. 2. Steady State Enzyme Kinetics
2.1 The Michaelis-Menten Equation
● Kinetic analysis of enzymatic reactions involving two or more substrates can be made easier by varying the
concentration of only one substrate at a time.
● Hence, the reaction rate will depend only on the concentration of the varying substrates.
● Assumptions:
➔The dissociation of E.P complex is not rate limiting.
➔The reversion of product to substrate is negligible.
➔When less than 5% of the substrate is consumed, the concentration of P is low.
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14. ● Michaelis-Menten equation relates the initial rate of an enzyme catalyzed reaction to the substrate concentrations and a ratio of
rate constants.
● It correlates velocity with substrate and enzyme concentration.
Where,
Total enzyme concentration [ET] = [E] + [ES]
Free enzyme concentration [E] = [ET] - [ES]
Substrate concentration = [S]
14

15. Initial velocity [V0] = Velocity measured immediately after mixing E + S, at the
beginning of reaction (initial velocity)
Maximum velocity = Vmax
Half Vmax = Km (substrate concentration)
● The initial velocity (v) is directly proportional to the enzyme concentration [E],
and that v follows saturation kinetics with respect to the substrate
concentration [S].
15

16. Michaelis-Menten equation explains hyperbolic curve:
1) At very low [S], {[S] << Km}
V0 approaches (Vmax/Km)[S]. Vmax and Km are constants, so linear relationship between V0 and [S].
1) When [S] = Km
V0 = ½ Vmax
1) At very high [S], {[S] >> Km}
V0 approaches Vmax (velocity independent of [S])
16

17. 2.2 Treatment of Kinetic Data
Analysis of Michaelis-Menten kinetics is greatly facilitated by a linear representation of the
data.
2.2.1 Lineweaver-Burk plot
Lineweaver-Burk plot suffers from the disadvantage that it emphasizes points at lower
concentrations and compresses data points obtained at high concentrations.
As a result it is not recommended for obtaining accurate kinetic constants.
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18. 2.2.2 Eadie-Hofstee plot
2.2.3 Hanes-Woolf plot
18

19. 3. Rapid, Reversible Inhibitors
● This class of inhibitors acts by binding to the enzyme’s active site in a rapid,
reversible, and noncovalent fashion.
● The net result is that the active site is blocked and the substrate is prevented
from binding.
● Types of rapid, reversible inhibitors:
3.1 Competitive inhibitors
3.2 Uncompetitive inhibitors
3.3 Non-competitive inhibitors
19

20. 3.1 Competitive Inhibitors
● It is structurally similar to those substrates whose reactions they inhibit.
● This means that a competitive inhibitor and enzyme's substrate are in direct competition for the same binding site on
the enzyme.
● The enzyme-bound inhibitor may either lack an appropriate functional group for further reaction, or may be bound in
the wrong position with respect to the catalytic residues or to other substrates.
● In any event, the enzyme-inhibitor complex E . I is unreactive (it is sometimes referred to as a dead-end complex)
and the inhibitor must dissociate and substrate bind before reaction can take place.
20

21. ki = Inhibition constant = Equilibrium constant for the dissociation of the enzyme-
inhibitor complex.
● The affinity of the substrate for the enzyme appears to be decreased in the
presence of a competitive inhibitor.
● Vmax = No change ; Km = Increased
21

22. 3.2 Uncompetitive Inhibitors
● Uncompetitive inhibitors do not bind to the free enzyme. They bind only to the
enzyme-substrate complex to yield an inactive E-S-I complex.
● Frequently observed in multisubstrate reactions
● Provides information about the order of binding of the different substrates.
● In a bisubstrate-catalyzed reaction, for example, a given inhibitor may be
competitive with respect to one of the two substrates and uncompetitive with
respect to the other.
22
Vmax = decreases; Km = No
change

23. 3.3 Noncompetitive Inhibitors
● Classical noncompetitive inhibitors have no effect on substrate binding and
vice versa, given that they bind randomly and reversibly to different sites on
the enzyme.
● They also bind with the same affinity to the free enzyme and to the enzyme-
substrate complex.
● Both E.I complex and E.S.I complex are catalytically inactive.
23
Vmax and Km both change.

24. Examples of Rapid, Reversible Inhibitors
1)
● This reaction is competitively inhibited by malonate (-OOCCH2COO-) that has,
like succinate, two carboxylate groups.
● It is therefore able to bind to the enzyme's active site but, with only one carbon
atom between the carboxylates, further reaction is impossible.
24

25. Examples of Rapid, Reversible Inhibitors (contd)...
2)
● SAH is a competitive inhibitor of SAM and a non-competitive inhibitor of
norepinephrine.
25

26. Classes of Reversible Inhibitors
26
Slow Binding Inhibitors Tight Binding Inhibitors Slow-Tight Binding Inhibitors
Takes considerable time to
establish equilibrium between E, I
and E-I complex.
Binds the target enzymes with
such high affinity that the
population of free inhibitor
molecules is depleted.
Tight binders with slow onset of
action are called slow-tight binding
inhibitors.
Slow on (association) rates Fast on rates Fast on rates

27. B] On the Basis of Structure/Mechanism
● An enzyme-catalyzed reaction must proceed from the ground state through a transition state
before products are formed.
● In addition, there are often some high-energy intermediates along the pathway.
● Knowledge and understanding of an enzyme's mechanism permits the identification of the
high-energy intermediates and the prediction of the structures of the transition states.
● It is possible to design enzyme inhibitors based on the structures of the various intermediates
along the reaction pathway.
● Inhibitors designed in this manner are occasionally referred to as mechanism-based
inhibitors.
27

28. 1. Ground State analogs
● The ground state of an enzymatic reaction consists of the substrates and the
products.
● Similar to the substrate analogs, product analogs can also be used to obtain
information about the binding mechanism of enzymes.
E.g.: Phosphonoformate (Antiviral agent used in the treatment of Herpes Simplex
Virus HSV and human cytomegalovirus HMV) acts as a product analog blocking
the pyrophosphate binding site, in the reaction catalyzed by DNA polymerase.
28
Phosphonoformate Phosphonoacetate

29. ➔ Initially, phosphonoformate and phosphonoacetate were identified as inhibitors of HSV DNA synthesis.
➔ Detailed kinetic studies, using DNA polymerase induced by avian herpes viruses, showed that phosphonoacetate
was a noncompetitive inhibitor of the four dNTPs.
➔ At low levels of dNTPs - Noncompetitive inhibitor;
➔ At saturating levels of dNTPs - Uncompetitive inhibitor;
➔ At very low Ki values - Competitive inhibitor of pyrophosphate.
➔ The inhibition patterns were identical to those observed using pyrophosphate as an inhibitor.
➔ Therefore it was concluded that phosphonoacetate acted as an analog of pyrophosphate and competed for the same
binding site.
➔ Later, both formate and acetate were confirmed as acting as pyrophosphate (i.e., product) analog inhibitors of
isolated HSV DNA polymerase.
29

30. 2. Multisubstrate Analogs
● A large number of enzymatic reactions involve the simultaneous binding of two or more substrates at the active site.
● There are two ways the two substrates, A and B, may bind to the enzyme to form an E.A.B complex.
● First, and most likely, they bind individually with dissociation constants of KA and KB.
● Second, the substrates may come together, positioned in such a way as to facilitate their subsequent reaction with a dissociation constant of
KBi.
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31. ● This reactive complex A.B then binds to the enzyme with a dissociation constant of KMS.
● In general, the formation of A - B is entropically unfavorable.
● However, a bisubstrate analog, designed to mimic A.B, can often be prepared by covalently connecting the corresponding
substrates or substrate analogs with a suitable linker group.
● Linking the two groups effectively overcomes the unfavorable entropic barrier.
● It has been calculated that an ideal bisubstrate analog inhibitor can bind up to 108 times more tightly than the product of the
substrate-binding constants.
● When two single-substrate analog inhibitors bind separately, but next to each other, two sets of translational and
rotational entropies are lost. However, when a bisubstrate analog inhibitor binds, it loses only a single set of
translational and rotational entropies.
31

32. ● Bisubstrate analogs binds to both the same sites as that of two single substrate analogs which results in gain in entropy.
● On the other hand, compared to the binding of a single-substrate analog, the multi-substrate analog inhibitor gains favorable binding enthalpies and
entropies from the additional binding site(s), while still losing only one set of translational and rotational entropies.
● Thus the binding of a multi-substrate analog should be very tight, without needing any assistance from transition-state complementarity.
● For the design of bisubstrate analogs, the two single-substrate inhibitors are then connected by an appropriate linker, and the optimal length of the linker is
determined experimentally.
● If enzyme binds substrate in a random manner:
then multi substrate inhibitor exhibits competitive inhibition with each substrates.
● If enzyme binds substrate in an ordered manner:
then inhibitor is competitive with first substrate and uncompetitive with other substrates.
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33. Advantages of multi substrate analogs:
➢ Provides high degree of specificity
➢ Combination of substrates usually produces unique structure
➢ Used for isozyme-specific inhibitors
E.g.: a] Glycinamide ribonucleotide transformylase (GAR TFase) catalyzes the
transfer of a formyl group from N10-formyltetrahydrofolate to glycinamide
ribonucleotide. This is a crucial step in de novo purine biosynthesis, which is
essential for cell division.
33

34. Bisubstrate inhibitor p-thioGARdideazafolate (P-
TGDDF) was synthesized by a thioether linkage and
was found to inhibit GAR TFase enzyme.
b] HMG CoA Reductase inhibitors (Mevastatin) are
present in the form of lactone. In vivo lactone is
converted to free acids. Mevastatin binds to the
hydroxymethylglutarate portion of the active site.
34
D,L-Mevalonate and D,L-3,5-hydroxy-
valerate, used as analogs of the upper
portion of the statins, were both poor
inhibitors, however, analogs of the
hydrophobic decalin region showed no
inhibitory effect.
Presumably, the upper portion of the
inhibitor is necessary for specificity and
the hydrophobic region for binding
affinity.

35. 3. Transition-state analogs
● The energy barrier imposed by the highest energy transition state controls the overall rate of the reaction.
● Enzymes bring about rate enhancements of 1010-1015 by lowering this energy barrier.
● They do this by having a greater affinity to the structure of the transition state than to the structures of either
substrates or products.
● Although an enzyme may have good affinity for its substrate, for E.S complex, the enzyme can further
stabilize the inherently unstable transition state by various methods.
● Simple transition-state theory states that the rate of an enzyme-catalyzed reaction is correlated with the rate
of a non catalyzed reaction by the same factor as the affinity of an enzyme for the transition state to the
affinity of an enzyme for a substrate.
35

36. ● The design of the transition state analog requires sufficient knowledge of the mechanism of the
target enzyme to predict the transition-state structure. Detailed knowledge of the existence of distinct
chemical steps, high energy intermediates, and their associated transition states are also useful.
● It is possible to design and synthesize an analog of a high-energy intermediate.
● Slow binding, tight binding, or structural similarity to the assumed transition-state structure are not, in
themselves, sufficient criteria to establish that an inhibitor is a true transition-state analog.
E.g. : Adenosine deaminase (ADA), which catalyzes the conversion of adenosine to inosine, is an
extremely proficient enzyme, providing a rate enhancement of more than 12 orders of magnitude.
36

37. Inhibitors of this enzyme have been used as immunosuppressants and are also
potential antitumor agents.
The enzyme-catalyzed reaction is thought to pass through an unstable hydrated
intermediate.
The potent inhibitors of this enzyme includes antibiotics coformycin and (R)-
deoxycoformycin.
The two antibiotics show at least 6-fold greater affinity for ADA than for the
substrate, suggesting that they are acting as transition-state analogs.
37
Coformycin (R)-deoxycoformycin

38. References
1) Abraham D.J.; Burger’s Medicinal Chemistry and Drug Discovery, 6th Edition,
Volume 1, 2003.
2) https://www.slideshare.net/KarthikReddy380/michaelis-menten-equetion
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