Enzyme Inhibitors

Inhibition of enzymes may be broadly classified under two categories-reversible and

irreversible inhibitors (Eq. 5.4). In the presence of inhibitor, the enzyme-substrate complex [E.S]
is replaced by [E.I] which may block or retard the formation of product. In the presence of a
reversible inhibitor, the enzyme is tied up and the reaction is retarded or stopped, however the
enzyme can be subsequently regenerated from the enzyme-inhibitor complex, [E.1], to react
again with substrate and produce product (see Eq. 5.3). On the other hand, irreversible inhibition
implies that the enzyme cannot be regenerated and the only way for catalysis to proceed would
be if new molecules of the enzyme are generated from gene transcription and translation.
Irreversible inhibition is commonly associated with covalent bond formation between inhibitor
and enzyme [E-I] which cannot be easily broken and is often defined as a time dependent loss of
enzyme activity. Reversible inhibition on the other hand does not necessarily imply non-covalent
bond formation. In many instances reversible inhibition can occur through covalent bond
formation, but these bonds can be hydrolyzed to regenerate free enzyme and inhibitor. Thus for a
reversible enzyme inhibitor there is no time
dependent loss of activity and enzyme activity can
always be recovered. There are instances when
reversible inhibition tends to look kinetically like
irreversible inhibition. This scenario results
whenever there is a tight binding of a reversible
inhibitor to the enzyme and consequently the
dissociation of the enzyme from this enzyme-
inhibitor complex is extremely slow. Kinetically it
is extremely difficult to distinguish this type of
inhibition from an irreversible inhibitor because
over time the enzyme does tend to look like it
loses its activity, and, for all practical purposes the
enzyme behaves as if it were irreversibly tied up.
In order to differentiate between tight binding
reversible and irreversible inhibitors, one can
dialyse the enzyme-inhibitor complex. In case of
the reversible inhibitor, on dialysis, the inhibitor
will be removed from the enzyme, resulting in
recovery of the enzyme activity; however, this is
not so with the irreversible one.

Irreversible Enzyme Inhibition

As previously described, irreversible enzyme inhibition is defined as "time dependent
inactivation of the enzyme" which implies that the enzyme has in some way or form been
permanently modified since it can no longer carry out its function. This modification is due to
the result of a covalent bond being formed with the inhibitor and some amino acid residue
present in the protein. Furthermore, this bond is extremely stable and for all practical purposes is
not hydrolyzed to give back the enzyme in its original state or structure. In most examples of
irreversible inhibition, a new enzyme must be generated through gene transcription and
translation for the enzyme to continue its normal catalytic action. Basically, there are two types
of irreversible enzyme inhibitors, the affinity labels or active site directed irreversible inhibitors
and the mechanism based irreversible enzyme inactivators.
Affinity Labels and Active Site Directed Irreversible Inhibitors
The affinity labels are those chemical entities that are inherently reactive and can target
any nucleophilic residue in the enzyme, especially those residing in and around the catalytic
center of the protein. These agents generally resemble the substrate so that they may bind in the
active site of the enzyme. In most examples, these agents also contain an electrophilic functional
group, which includes groups such as: halo-methyl ketones (X-CH2C=O, where X = halide),
sulfonyl fluorides (S02F), nitrogen mustards ((ClCH2CH2)2NH), diazoketones (COCHN2), and
other such reactive groups, that can "label" or alkylate a nucleophilic amino acid residue present
in the enzyme. They generally tend to be indiscriminate in their action and have little therapeutic
value since they are non-selective and thus inherently toxic. They have been used mainly as
biochemical tools to probe active sites of enzyme so as to discern the types of amino acid
residues present in and around the catalytic center of an enzyme.
The classic example of an affinity label is TPCK
(tosyl-phenylalanyl-chloromethyl-ketone), an irreversible
inhibitor of the serine protease, chymotrypsin. Since
TPCK resembles the amino add phenylalanine, it can
bind to the active site of the chymotrypsin whose
selectivity is for such hydrophobic amino acid residues
(Phe and Tyr). In the course of normal peptide hydrolysis, the reactive chloromethyl-ketone
labels the nucleophilic histidine residue present as part of the catalytic triad (Ser-His-Asp) in the
active site of the protease. Another similarly designed affinity label is TLCK (tosyl-lysy1-
chloromethyl-ketone) whose specificity is for the protease trypsin. Trypsin cleaves peptide bonds
adjacent to the basic amino
acids, lysine and arginine. It
was found that TLCK was a
specific inhibitor of trypsin
but had no activity for
chymotrypsin. On the other
hand TPCK while extremely
specific for chymotrypsin
showed no activity for trypsin.
Because of the inherent reactivity and non-selectivity of these affinity labels and their
limited utility in drug therapy, the late B.R. Baker extended this concept to design inhibitors that
would have greater selectivity and specificity and thus be potential drug candidates. He designed
several analogues, termed active site directed irreversible inhibitors, targeted towards
thymidylate synthase, a key enzyme
involved in the de novo metabolism
of thymidylate. These analogues
contained a substrate binding region
linked to a reactive group such as a
halomethyl ketone by a tether whose
chain length could be manipulated.
The substrate portion of the
analogue ensures both affinity and
rapid binding to the enzyme active
site. Once bound, areas in and
around the binding site and on the
surface of the enzyme could be
probed for nucleophilic amino acid residues. By manipulating the length of the tether, ideal
inhibitors could then be designed such that any suitably located, sufficiently nucleophilic amino
acid residue, present on the surface of the enzyme could potentially be alkylated by the
halomethyl ketone (Fig). Once alkylated, the tether "bridges" the active site with the labeled
amino acid residue thus "tying up" and preventing further catalysis by the enzyme.

Mechanism-Based Irreversible Enzyme Inactivators

The mechanism-based irreversible inhibitors have also been termed as "suicide
substrates, "kcat inhibitors," "Trojan horse inhibitors" or "latent alkylating agents." These
inhibitors are inherently unreactive but upon normal catalytic processing by the enzyme, are
activated into highly reactive moieties. These reactive functionalities can then irreversibly
alkylate a nucleophilic amino acid residue or cofactor present in the enzyme and in essence,
cause the enzyme's death ("suicide"). Basically, these inhibitors have a latent reactive
functionality that only becomes apparent after binding and acted upon by the normal catalytic
machinery of the enzyme. This type of inhibitor design differs from the preceding one in that
these inhibitors have one more level of selectivity built in to them. The kinetic scheme for such
inhibition is shown in Equation 5.5, where enzyme, E, binds with substrate (inhibitor) S to give
an [E . S] complex with dissociation constant of Ki. Next the [E . S] complex is converted into a
highly activated complex [E . S*] by the catalytic machinery (kcat) of the enzyme, which can then
go on to alkylate the enzyme, [E-S]. Note that it is possible for the reactive species [S*] to
diffuse (dissociate) from the enzyme and
react with some other target (nucleophilic
species), i.e., the system is "leaky;"
however, if this happens then the inhibitor
cannot be classified as a "true" suicide
substrate because specificity is lost.
There are several requirements that need to be met by these inhibitors in order for them to be
classified as suicide substrates. These include: inactivation should be time dependent (reaction
should be irreversible); kinetics should be first-order; the enzyme should show saturation
phenomenon; the substrate should be able to protect the enzyme; stoichiometry of the reaction
should be 1: 1 (one active site to one inhibitor).

Examples of Suicide Substrates. Halo Enol Lactones.

Halo enol lactones are an example of suicide inhibitors for serine proteases. These
analogs were developed by Katzenellenbogen and coworkers at the University of Illinois. Upon
normal catalytic processing by
the serine hydroxyl
functionality, they give rise to a
reactive halo-methyl ketone
which subsequently alkylates a
nearby nucleophilic residue on
the enzyme (Fig).

Reversible Enzyme Inhibition

Reversible enzyme inhibition may be classified under two main headings, competitive and non-
competitive. Competitive inhibition, by definition, requires that the inhibitor competes with the
substrate for binding to the enzyme at the active site and this binding is mutually exclusive. That
is to say that if the inhibitor binds to the enzyme the substrate will not be able to bind and vice-
versa. But, competitive inhibition also suggests that the inhibition can be reversed in the presence
ofsaturating amounts of substrate, since in this case all enzyme active sites will be occupied by
substrate displacing inhibitor. In contrast, non-competitive inhibition implies independent
binding, i.e., both inhibitor and substrate may bind to the enzyme at different sites. Since binding
of the inhibitor to the enzyme is at a site other than the active site, non-competitive inhibition
cannot be reversed by increasing the concentration of substrate.
Most of the rationally designed and clinically useful reversible inhibitors are competitive
inhibitors. These inhibitors generally bear some structural resemblance to the natural substrate of
the enzyme. The design of such inhibitors would thus seem a logical and rational task which is
uniquely suited to the medicinal chemist who can utilize the principles of bio-isosteric
modification of natural enzyme substrates and metabolites, or modification of "lead" structures
and structure activity relationships, to create selective and potent inhibitors. However, there are
pitfalls in this endeavor since even the most rationally designed drug must still overcome
transport and other cellular barriers before exerting its effects. In the case of the non-competitive
inhibitors, the design is not as straightforward. These inhibitors can have widely differing
structures which in many instances bear no resemblance to the natural substrate. In general,
inhibitors of the non-competitive type have been primarily obtained through random screening of
chemically novel molecules followed by further synthetic manipulation of the pharmacophore to
optimize their inhibitory effects.

Examples of Reversible Inhibitors

The design of enzyme inhibitors has included random screening of synthetic chemical
agents, natural products and combinatorial libraries followed by molecular optimization or
structure activity relationships of so called "lead" structures as well as bio-isostere analogs of the
enzyme substrates themselves. Drugs have also been developed for one indication but based on
observed side-effects have lead to other uses . The rational approach in the design of enzyme
inhibitors is greatly aided if the enzymatic reaction is characterized in terms of its kinetic
mechanism. Such a characterization would include the knowledge of the kinetic parameters (rate
constants and dissociation constants) of individual steps in the overall reaction pathway, as well
as, the characterization of (any) intermediates involved in these individual steps. Examples of
such "rational" inhibitors include both the reversible and irreversible inhibitors of enzymes.

Inhibition of Acetylcholinesterase
Acetylcholinesterase (AChE) is the enzyme that catalyzes the catabolism of the
neurotransmitter acetylcholine to acetate and choline. Thus, inhibition of AChE would lead to
increased concentrations of
acetylcholine and a prolonged action
of the neurotransmitter. Inhibitors of
AChE have found use in cases of
glaucoma and Alzheimer's disease. In
order to appreciate the design of these
inhibitors it is useful to first
understand the mechanism of action of
AChE. AChE has an anionic site
which can bind the positively charged
quaternary ammonium group of the
choline functionality and an active
esteratic site which contains a
nucleophilic serine residue involved in the hydrolysis of the ester bond (Fig). The mechanism
involves the attack of the nucleophilic serine hydroxy group on the carbonyl group of
acetylcholine to form a tetrahedral intermediate which breaks down resulting in the release of
choline and an intermediate, acetylated serine which subsequently hydrolyses to release AChE.

Physostigmine has been used in the

treatment of glaucoma. It is an alkaloid
with a carbamate moiety which resembles
the ester linkage of acetylcholine. Being an
alkaloid it is protonated at physiological
pH, and thus can bind to the anionic site of
AChE. Following the mechanism of AChE
the serine residue of the enzyme can attack
the carbonyl group of physostigmine and in
the process the serine is carbamylated
(Fig). This carbamyl serine intermediate is
stable and subsequent hydrolysis by water occurs extremely slowly. The carbamylated enzyme is
only slowly regenerated with a half-life of 38 min., more than seven orders of magnitude slower
than that for the natural substrate, acetylcholine. This is an example of a reversible inhibitor
which is involved in covalent bond formation with the enzyme which ultimately gets hydrolyzed.

Inhibition of Human
Immunodeficiency Virus-Reverse
Transcriptase (HIV-RT)
Azidothymidine (AZT). The advent
of AIDS stimulated a great interest
in designing inhibitors against the
essential viralpolymerase-HIV-RT.
AZT is a potent inhibitor of HIV-
RT, the retroviral polymerase which
catalyzes the for- mation of proviral
DNA from viral RNA. AZT is
structurally similar to the natural
nucleoside thymidine but has an
azide group (-N3) rather than a
hydroxy group (-OH) at the 3'-
position of the sugar, deoxyribose
(Fig). AZT is activated intra-
cellularly to its triphosphate and
competes with thymidine
triphosphate for uptake by HIV-RT
into DNA. Once incorporated,
further chain extension of the DNA
is prevented since there is no 3'-
hydroxyl group to continue the DNA
synthesis. In this fashion, AZT is an
effective chain terminator of viral
DNA synthesis.