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ab
c
. • Receptors as Target Structures for Drugs
. Fig. 22.6 a The nicotinic acetylcholine receptor (nAChR) is ali-
gand-gated ion channel (Sect.30.4). Here the cylinders do not stand
for segments but rather for ve separate proteins, each of which has
four transmembrane domains. After binding acetylcholine, the channel is quickly opened. For example, chloride channels use the same
principle of construction (Sect.30.7). bSoluble receptors dimerize after agonist docking to their ligand-binding domains (LBD). Here homodimers composed of two identical receptors as well as heterodimers
are known, but new ones are still being discovered and
characterized (Sect.29.1).
After an agonist docks, the active conformation of
the receptor is stabilized. Antagonists prevent agonists
from docking, and inverse agonists stabilize the inactive
conformation of the receptor.
The initiated receptor response proceeds along identical pathways, despite the different receptor types, and
then branches off again. This economical principle of
Nature is also used in other cases, such as the regulation
of cell proliferation. The more or less pronounced effect
specicity is achieved by
The different structures of the agonists and receptors
-
and the resulting activation of different G-proteins
and effector proteins,
The different receptor occupancy and density of dif-
-
ferent cells, and
The location of the cells that produce and release the
-
hormone or neurotransmitter. This is accomplished
in very specic cells; neighboring cells or organs are
not involved.
of two different receptors can be formed. The so-called zinc ngers of
the DNA-binding domains (DBD) recognize very specic sequences
of DNA. Aparticular DNA segment is addressed by dimerizing two
receptor units. cMembrane-bound receptors for growth factors and
insulin also dimerize. Two receptors form acomplex in the membrane
and in doing so activate the intracellular domain of the receptor, in
this case, atyrosine kinase
The picture of such receptors can be very complex. For
example, there are two distinct groups of acetylcholine re-
ceptors that preferentially bind either muscarine, atoxin
from the toadstool Amanita muscaria, or nicotine, the
active ingredient in the tobacco plant Nicotiana tabacum.
Unlike the muscarinic acetylcholine receptor, the nicotinic acetylcholine receptor (nAChR) is aligand-gated
ion channel (Sect.30.5). It has acomplex architecture
of ve protein chains located in the cell membrane
(. Fig.22.6a). For the nAChR from the electric organ of
the torpedo electric ray, ash, cryo-electron microscopy
images are available of the closed and open structure (after activation by acetylcholine) of the 290 kD nAChR
protein complex (Sect.30.5).
Many hormone receptors, such as those for thyroid
hormone, sex hormones, the corticosteroids, and retinoic
acid, are soluble receptors that can move freely in the cytosol, or cell uid. After binding the agonist, the complex
translocates to the nucleus. There it binds as adimer to
the signal sequences of the DNA, operator and repressor genes and induces or suppresses the new synthesis
Chapter • How Drugs Act: Concepts for Therapy
22
of specic proteins (. Fig.22.6b). These receptors are,
therefore, also referred to as transcription factors.
All cytosolic hormone receptors or nuclear receptors
are based on common structural principles (Sect.28.2).
They have domains with aDNA-binding site and a ligand-binding site. The DNA-binding site is highly conserved, i.e., its amino acid sequence varies very little
between different receptors. It contains two “zinc ngers” consisting of two Zn2+ binding sites, which are
highly conserved motifs that bind to very specic DNA
segments known as recognition sequences. The ligand
binding site is much more variable. Dimers, either of
two identical receptors (homodimers) or of two different receptors (heterodimers), are formed to interact with
DNA. Four zinc ngers in the dimer recognize atotal of
12base pairs of DNA.
Dimerization is also found in other classes of membrane-bound receptors that are not GPCRs. These include receptors for growth factors such as human growth
hormone (hGH), epidermal growth factor (EGF), and
insulin (Sect.29.8). Upon binding to the factor, these
receptors dimerize with the extracellular domains in the
membrane. This activates intracellular kinases that are
part of the receptor protein (. Fig.22.6c). In addition,
there are receptors that must form complexes of more
than two units to elicit areceptor response. These include anumber of immunologically important receptors,
as well as receptors for nerve growth factor (NGF) and
tumor necrosis factor (TNF, Sect.29.8).
Several examples of proteins that function as oligomers are presented in this section. In fact, oligomerization
is also common in enzymes. There are many reasons why
oligomerization is advantageous. On the one hand, there
are functional requirements that require multiple adjacent domains, as described above. On the other hand,
there may be mechanistic advantages, especially for enzymes. Individual domains of an oligomer are not necessarily independent. Their catalytic efciency may depend
on the current state of the other domains of the oligomer. This provides an additional means of regulating
protein function. Oligomerization can also have another
signicance. The interior of acell is highly crowded with
proteins, ligands, substrates, and ions. It can be compared to aticker-tape parade given to awinning football
team: hectic pushing and shoving! One way to reduce this
number without compromising catalytic productivity by
sacricing catalytic centers is to form oligomers.
22.6 Drugs Regulate Ion Channels:
Our Extremely Fast Switches
Ion channels embedded in the cell membrane allow ions
to enter or leave the cell along an electrochemical gradient when open. The opening or closing of the channel
can be either voltage-, ligand-, or receptor-gated. All of
these processes are extremely fast (Sect.30.1).
Many drugs act on voltage-gated ion channels
(Sect.30.4). Local anesthetics and their derivatives are
sodium channel blockers; they reduce the excitability of
nerve cells. The fugu sh toxin, tetrodotoxin (Sect.30.4),
also blocks this channel. Other antiarrhythmic drugs
block potassium channels. Substances that stabilize the
K+ channel in the open form, called K+ channel openers,
have vasodilatory and antihypertensive effects. The antidiabetic sulfonylureas are K+ channel blockers that act on
the insulin-producing cells of the pancreas (Sect.30.2).
Voltage-gated calcium channels are modulated by ligands
such as the nifedipine-type antagonist or blockers such
as the verapamil or diltiazem-type ligands (Sect.30.4).
The nicotinic acetylcholine receptor (nAChR,
. Fig.22.6a), glutamate receptors, and GABA recep-
tors belong to the Cys–loop family of ligand-gated ion
channels (Sects.30.5, 30.6, and30.7). The opening and
closing of the channel is not caused by an electrical impulse, but by the binding of aligand.
Benzodiazepine-type tranquilizers (Sect.30.7) enhance
the binding of the neurotransmitter g-aminobutyric acid
(GABA) to chloride channels. Prolonged opening of this
channel results in an increased inux of chloride ions
and achange in the response behavior of nerve cells.
They bind between different domains of the channel.
Barbiturates and inhaled anesthetics also act on GABA
receptors, but at adifferent binding site between different
domains.
22.7 Blocking Transporters
and Water Channels
Transporters are proteins that affect the active uptake of
molecules or ions into cells. They play acrucial role in the
digestive process. Because amino acids and sugars cannot cross membranes on their own, they can only be absorbed in the digestive tract with the help of transporters.
Transporters are also extremely important for signal
transmission in nerve cells. Once released, aneurotrans-
mitter must be quickly removed from the synaptic cleft
to prevent prolonged stimulation of the nerve cell. This
is accomplished in part by metabolic degradation, but
this is very wasteful for the releasing cell. Uptake (often
incorrectly called reuptake) by aspecic transporter is
more economical. The neurotransmitter is stored in vesicles and made available for the next release.
Transporters work against concentration gradients.
The transport process is relatively slow, much slower
than an ion channel, and it requires energy. The amino
acid sequence of the specic transporter is known for
many neurotransmitters, amino acids, sugars, and nucleosides. Like G-protein-coupled receptors, transport-
. • Modes of Action: ANever-Ending Story
ers are divided into many families. Most have an even
more complex structure with 12transmembrane domains
(Sect.30.10).
Afew drugs target the transporters directly and displace the natural ligands. The euphoric effects of cocaine (Sect.3.4) are due to its binding to the dopamine
transporter, which is responsible for the active transport
and uptake of dopamine into neurons. Arapid onset
of cocaine causes adelayed uptake of dopamine from
the synaptic cleft, which is responsible for the typical
physical and psychiatric effects. Some antidepressants
are ligands for the noradrenaline and serotonin transporters (Sect.1.4). They are bound but not transported
into the cell. In contrast, some amino acid analogues
are transported into neurons by transporters and act as
neurotoxins. The complex interplay of neurotransmitters, enzymes, receptors, and transporters is illustrated in
. Fig.22.7. Some anti-gout drugs bind to the uric acid
transporter. They displace uric acid, inhibit its absorption from primary urine, and accelerate its excretion with
urine. There are even specic transporters for bile acids.
In addition to the transporters described above, other
members of this protein class are also important for the
uptake or excretion of foreign substances into or from
cells. Tumor cells often respond to therapeutic measures
by developing multiple resistance to many structurally
different substances (Sect.30.10). The glycoprotein GP
170, also atransporter with 12transmembrane domains,
is responsible for this process.
In contrast to ion channels, ion transporters work
against concentration gradients. This is an active process
that requires energy (Sect.30.9). Drugs can also affect
this process. One example is drugs that increase urine
production: diuretics. They inhibit different ion transporters. The Na+/K+ ATPase, apump that exchanges
sodium for potassium ions, is inhibited by cardiac glycosides, which are used to treat heart failure. Substances
such as omeprazole (Sects.9.5 and30.9) inhibit the H+/
K+ ATPase, the so-called proton pump. Nature uses spe-
cial water channels to regulate water homeostasis and
also to transport small, uncharged molecules such as
glycerol or urea across the cell membrane quickly and
selectively. Unlike transporters, and analogous to ion
channels, these aquaporins allow water to ow along the
osmotic gradient (Sect.30.12). Ten isoforms with different permeabilities have been discovered in mammals.
They are tetramers composed of six transmembrane helices. Each monomer unit forms achannel. The channels
are partially made available for water homeostasis by the
release from cytosolic vesicles or they can be activated by
phosphorylation. Regulation of the water channels by
drugs represents aconcept for diuretic therapy, but the
treatment of parasitic infections has also been discussed
as an additional indication.
22.8 Modes of Action: ANever-Ending
Story
Therapies for viral, bacterial and parasitic diseases attempt to target aspecic pathogen. Different mechanisms are used, such as biosynthetic pathways that are
not present in humans in identical form or that do not
play an important role in humans. In this way, the risk of
adverse effects can be minimized from the outset.
Antimetabolites are substances that are incorporated
as afalse substrate instead of the natural biological reagents, for example as enzyme cofactors or in DNA. One
example is the sulfonamide sulfonamidochrysoidine. Its
cleavage product sulfanilamide (Sect.2.3) is similar to
p-aminobenzoic acid, which is the starting material in
the biosynthesis of an important bacterial cofactor, dihydrofolic acid. Only bacteria are affected. Humans are
not dependent on this biosynthetic pathway. Like other
. Fig. 22.7 Nerve signal transmission through neurotransmitters is
based on acomplex interplay of enzymes, receptors, ion channels, and
transporters. Dopamine is produced by enzymatic decarboxylation of
the amino acid l-DOPA. As with other neurotransmitters, it is stored
in special vesicles. Upon electrical stimulation, Ca2+ ions ow into the
cell. This causes the neurotransmitter to be released into the synaptic
gap. The nerve impulse is conducted further by the interaction with
the postsynaptic receptor. Finally, the uptake in the presynaptic cell
is accomplished by atransporter and the neurotransmitter is stored
in a vesicle again, or degraded by the enzyme monoamine oxidase
(MAO). In the case of dopamine deciency, dopamine can be released
by enzymatic decarboxylation when the drug l-dOPA, an amino acid
that enters the nerve cell through the amino acid transporter, is administered
Chapter • How Drugs Act: Concepts for Therapy
22
mammals, humans must obtain dihydrofolic acid from
the diet. Some antiviral and anticancer drugs are nucleo-
side analogues. Depending on the type of structure, they
use amodied base, amodied sugar, or both. All affect
DNA or RNA synthesis. Acyclovir and afew other analogues enter cells as Trojan horses in an inactive form
and are “armed” once inside the cell. Their activation is
carried out by viral enzymes, and this process occurs only
inside cells that have been infected by the virus (Sects.9.5
and32.5). Another mechanistic principle attempts to interfere with the translation process so that certain proteins are never produced by protein biosynthesis. The
translation of mRNA is blocked by complexation with
antisense oligonucleotides (Sect.32.4). The resulting dou-
ble-stranded mRNA cannot be read by the ribosome.
This type of therapy can be used to treat exaggerated
immune reactions, septic shock, arterial hypertension,
pulmonary emphysema, or pancreatitis.
Many antibiotics, such as the penicillins and cephalosporins (Sect. 23.7), inhibit bacterial cell wall bio-
synthesis. In the latter process, they block the catalytic
center of atranspeptidase, which has asimilar mode of
action to aserine hydrolase (Sect.23.7). The antibiotic
d-cycloserine, also an inhibitor of cell wall construction,
penetrates into the interior of the bacteria using ad-alanine transporter. Other antibiotics are inhibitors of
protein biosynthesis (Sects.32.6 and32.7). Tetracycline
(Sect.6.4), streptomycin (Sect.6.4), and chloramphenicol (Sect.9.2) also inhibit the protein synthesis machinery. They interact with the 30S or 50S subunit of the
ribosome and block ribosomal peptide synthesis. The
elucidation of the spatial structure of the ribosome has
provided abasis for understanding the mechanism of
action of alarge number of macrolide antibiotics and
has provided aperspective on the development of resistance mechanisms (Sect.32.7). Antibacterial quinolone
carboxylic acids inhibit gyrase. Gyrase is an enzyme that
causes DNA to twist, thus, allowing DNA to be tightly
packed in bacterial cells. Without this twisting, there is
simply not enough space in the cell for the genetic material (Sect.32.6). Polyene antibiotics are used to treat
fungal infections. They form channels in the fungal cell
membrane that cause aloss of intracellular ions and,
consequently, cell death. Azoles inhibit the biosynthesis
of ergosterol, which is essential for the formation of an
intact cell membrane.
Alkylating agents play an important role in tumor
therapy. Alkylation of DNA bases results in reading and
writing errors that are much more severe in rapidly dividing tumor cells than in normal cells, but also have
signicant side effects (Sect.12.14). Intercalating tumor
therapeutics are planar molecules that slip between two
base pairs of DNA (Sect.14.9). The resulting disruption also leads to errors in cell division. Other DNA ligands bind to the minor or major groove on the outside
of the double helix. Paclitaxel (Taxol, Sect.6.1) and the
epothilones are important agents in cancer therapy. They
bind to tubulin, aprotein that forms tube-like structures
called microtubules. Since the formation of microtubules
is essential for cell division, paclitaxel and the epothilones
inhibit this process in avery specic way.
The immunosuppressant cyclosporine (Sect. 10.1,
. Fig.10.2) blocks the activation of the immune system,
the so-called helper cells. Two enzymes are involved in
this process. One, cyclophilin, is aprolyl cis–trans isom-
erase. The other, calcineurin, is aCa2+/calmodulin-dependent phosphatase. Cyclosporine acts as a“putty” (or
molecular glue) between these two proteins. The complex
prevents the activation of helper cells and, thus, stops
the stimulation of an immune response. Modern transplantation surgery would not be possible without the immunosuppressive cyclosporine and substances with an
analogous mode of action.
RAS proteins play an important role in tumorigenesis. They are afamily of relatively low molecular weight
enzymes. RAS proteins with mutated active centers
lose their ability to control cell division, and the cells
divide unstoppably. They are therefore oncogenic, i.e.,
they cause tumors. About 50% of all lung and colon
tumors have mutated ras genes, and about 95% of the
ras genes in pancreatic tumors are mutated. There are
other approaches to therapy. RAS proteins must move
from the cytosol, the cell uid, to the cell membrane to
signal cell division. To do this, they are enzymatically
tagged with afarnesyl group that anchors the protein to
the cell membrane. Preventing membrane anchoring by
inhibiting farnesyl transferase is an attractive approach
for targeted cancer therapy (Sect.26.11). It has now been
shown that this principle of blocking protein farnesylation can also be used to treat parasitic infections. The
farnesyl transferases of these parasites are the targets for
drug development.
Tumor suppressor genes produce proteins, such as
the p53 protein, that prevent cell division in the event of
DNA damage. Any genetic defect in acell that results in
reduced levels of one or more of these proteins will allow
cells with defective DNA to proliferate. Cell division becomes uncontrolled, and atumor forms with additional
genetic defects and uncontrolled growth.
Vascular occlusion is caused by the aggregation of
platelets. Cell surface proteins called integrins, such as
the adhesion glycoprotein α
, play an important role.
IIbβ3
Two of these molecules form acomplex with brinogen
that “glues” cells together. The targeted development of
small peptidomimetics (Sect.10.6) based on an RGD
(Arg–Gly–Asp) motif has been amajor success in rational drug design (Sect.31.2). Another system that plays
an important role in cell–cell recognition between leukocytes and endothelial cells are the selectins. In cases
of inammation, E- and P-selectins are upregulated
and presented on the endothelium, preventing leukocytes from rolling along the surfaces of the blood vessels
. • Resistance and Its Origin
(Sect.31.3). After adhesion, the leukocytes enter the vessel and migrate to the site of inammation to ght the infection. In some diseases, excessive leukocyte inltration
leads to tissue damage. To prevent this, attempts have
been made to interfere with the inammatory cascade
with compounds that block the surface expression of selectins. These receptors recognize sugar-like molecular
groups on the surface of leukocytes, so the development
of appropriate carbohydrate-based antagonists represents
asuitable therapeutic concept.
For infection to occur, asurface contact must be established between the inuenza virus and the host cell.
The virus docks to the host cell with its capsule protein,
hemagglutinin, to initiate endocytosis. Once inside the
cell, the virus uses the protein biosynthetic machinery of
the infected cell to make copies of itself. After maturation, the new virus must be expelled from the cell. To do
this, the new virus buds on the cell surface and the bud
is then snipped off. In the nal step, viral neuraminidase
cleaves sialic acid. This acid binds the viral hemagglutinin
to the host cell. This nal step can be blocked by neuraminidase inhibitors (Sect.31.5). The inhibitors zanamivir
and oseltamivir have been very successful. The CCR5
receptor antagonist maraviroc has been introduced for
the treatment of human immunodeciency virus (HIV)
infection; the CCR5 receptor acts as an entry gate for the
human immunodeciency virus and its inhibition blocks
host cell invasion.
The body’s immune system has developed very effective defense mechanisms. Antibodies are one such defensive weapon. These proteins are able to selectively bind
to foreign substances with high afnity and present them
to phagocytic cells (i.e., dendritic cells and macrophages)
for degradation. This sophisticated, highly specic recognition system for molecules ranging from very small
low molecular weight antigens to complex macromolecular systems has been tapped for pharmaceutical therapy
(Sect.32.3). Today, about 200 articially produced antibodies against awide variety of targets are used in the
treatment of many different diseases. There is no end in
sight, as many newly developed antibodies are currently
in clinical trials.
There are very few truly “nonspecic” drugs. Antacids, which neutralize gastric acid purely chemically, belong to this class, as do purely surface-active substances
such as amphiphilic bactericides, fungicides, and hemolytics. Even the barbiturates, local anesthetics, inhalation
anesthetics, and alcohol, long thought to be anonspecic
agent, have been shown to have specic mechanisms of
action. Frequently, the demonstration of aspecic effect
has been made through the differential effects of pure
enantiomers of aracemate. The β-antagonistic effect of
an optically active β-blocker is associated with one enantiomer (Sect.5.5). However, the nonspecic adverse
effects on membranes are attributed equally to both enantiomers.
Is there anything new to discover? An absolute surprise was the discovery that nitric oxide(NO), atiny gaseous molecule, is also aneurotransmitter. Substances
that release NO or interfere with NO biosynthesis lower
or raise blood pressure (Sect.25.8). New subtypes of
established receptors are constantly being discovered.
The extent to which it makes sense to optimize acompound for absolute receptor specicity remains unsolved.
It may well be the case that some compounds targeting
multiple receptors or their subtypes are better suited for
therapy than highly specic analogues. This is particularly true for compounds that bind to GPCRs. Here,
the activity prole against awhole range of receptor
subtypes is critical for the efcacy of acompound. Numerous GPCRs are even involved in our sense of smell,
which follows this principle of multiple graded receptor
responses (Sect.29.7). This is the only way to achieve
the nely tuned and nuanced diversity of perception.
This is abroad area of research. To date, especially with
CNS agents, only clinical research can provide the results
needed to make adecision about the therapeutic utility
of acompound.
22.9 Resistance and Its Origin
Pathogenic viruses, bacteria, and parasites evolve resistance to drug therapy. Inappropriate and excessive use
of antibiotics in the past has led to selection pressure for
resistant strains. Unfortunately, hospitals are the primary
site for the emergence and spread of resistant strains. The
spatial proximity and concentration of different pathogens is virtually unavoidable. In some cases, there are
only afew effective weapons left, such as glycopeptide
antibiotics. They should be used prudently and purposefully, even if this goes against the commercial interests
of the manufacturer.
Most bacterial pathogens defend themselves against
penicillins and cephalosporins by producing β-lact-
amases (Sect.23.7). These are enzymes that open the
four-membered lactam ring of these antibiotics to inactive cleavage products. During the long period of optimization of this class of substances, metabolically stable
analogues as well as specic β-lactamase inhibitors have
been developed.
The retrovirus HIV, which causes the immune deciency disease AIDS (Sects.1.3 and24.3), transfers its
genetic information from RNA back into DNA. This
process is aficted with an extremely high error rate of
about one base mutation per generation. The high mutation rate leads to the rapid emergence and selection
of resistant strains. In the last decade, many drugs with
completely different modes of action against HIV have
been introduced to the market, but resistances to many
inhibitors have been observed very quickly, for example,
against the HIV protease (Sect.24.5) or reverse transcrip-
Chapter • How Drugs Act: Concepts for Therapy
22
tase inhibitors (Sect.32.5), and even multiple resistances.
The mutated viruses are even resistant to several structurally different inhibitors! Combining different drugs
against the same target is of little help here. Only acombination of compounds that hit the virus at completely
different instances of its life cycle offers areprieve.
Tuberculosis is also re-emerging. Resistant pathogens
require the development of new therapeutics. After the
convincing success of mosquito control with DDT and
therapy with synthetic antimalarials, malaria is making
acomeback in developing countries (see Sect.3.2).
The major problem in anticancer therapy is the development of multidrug resistance (MDR) during treatment.
The resistance is not only to the anticancer drug, but
also to several other anticancer drugs. This multidrug
resistance is due to the overexpression of a transporter
(Sect.30.10), glycoprotein 170, which can largely elimi-
nate structurally different xenobiotics from the cell. Although GP170 prefers cationic substances, another transporter, multidrug resistance-associated protein (MRP),
eliminates amphiphilic anionic substances, i.e., compounds with polar and nonpolar character. However,
amphiphilic substances are also capable of breaking the
resistance of tumor cells. Quantitative structure–activity relationships show that the resistance of tumor cells
to certain drugs is mainly associated with similarities
in their molecular weights, i.e., the size of the inducing
agent, and its lipophilicity.
22.10 Combined Administration of Drugs
Combination drugs are popular with pharmaceutical
manufacturers, physicians, and patients alike. Manufacturers like them because they expand the indications for
asuccessful drug and boost sales. Some physicians like
the fact that therapy is simplied in many cases, while
others oppose such combination products. One advantage
for older patients is that they no longer have to take so
many different drugs at different times of the day and in
different doses, but only one or afew combination drugs.
This improves compliance, because one of the most common reasons for treatment failure is patient behavior. Either the regular dose is forgotten, or the patient takes
abreak from the regimen over the weekend or while on
vacation. These behaviors are particularly pronounced
in elderly patients, with medications that do not have an
obvious immediate effect, or with medications that have
side effects that the patient subjectively perceives as unpleasant.
Clinical pharmacologists, academics, and many critically minded physicians have considerable reservations
about combination products. This is understandable
when one considers that apatient’s attitude to aparticu lar drug requires the observation of adose–response
relationship over along period of time and, ultimately,
individualized therapy. In acombination drug, there is
always axed relationship between the individual components. Many combinations, such as analgesics, contain
components with different modes of action. These are
often misused without astrict medical indication and
must, therefore, be judged critically.
There are reasonable combinations that even opponents to the general concept of combination therapy
would accept without reservations. Among these are
l-DOPA preparations with which the side effects can
-
be reduced by selective combination (Sects.9.4, 26.10,
and27.8),
Antihypertensives and diuretics, the different mecha-
-
nistic principles of which complement each other,
Antibacterial preparations in which adihydrofolate
-
reductase inhibitor (Sect.27.2) is combined with an
appropriate sulfonamide,
Hormonal contraceptives (Sect.28.5), and
-
Polyvalent vaccines, with which asingle application
-
offers protection against multiple diseases.
In the case of l-DOPA therapy, only combinations of
several agents reduce side effects to atolerable level. In
the case of antihypertensives and diuretics, asingle principle is often not sufcient to achieve the same effect as
combinations. In the case of sulfonamide combinations
and antituberculosis compounds, multiple modes of action can prevent or delay the development of resistance.
An inhibitor of the P450 family of metabolic enzymes
may be justied as an adjunct to expensive drugs or to
drugs used at very high doses. In this way, the concentration of the other drug can be maintained at ahigher
level and for alonger period of time (Sect. 27.7). An
important prerequisite for all combination drugs is an
adequate therapeutic window and adapted pharmacokinetics of the components, at least those that support the
actual mode of action.
22.11 Synopsis
Arelatively small portion of the druggable genome
-
has been pharmaceutically addressed, and GPCRs
overrepresent the targets for which active substances
are available. Protein kinases represent aparticularly
promising emerging family of targets.
Enzymes are very popular drug targets, and the natu-
-
ral substrates often provide the starting point for ara-
tional drug design approach. There are three types of
enzyme inhibitors: competitive inhibitors, noncom-
petitive inhibitors, and allosteric inhibitors. Enzyme
inhibition can also be classied as reversible and irre-
versible. Nowadays reversible inhibition is desired, but
some very important drugs are irreversible inhibitors,
and some reversible inhibitors have such high afnity
that they are de facto irreversible inhibitors.
Bibliography and Further Reading
Receptors are also important drug targets; they can
-
be subdivided into GPCRs, ion channels, hormone
receptors, and growth factor receptors. An agonist activates the receptor, an antagonist prevents the agonist
from docking at its binding site, and an inverse agonist
stabilizes an inactive conformation of the receptor.
Ion channels are extremely fast gateways for ions
-
and can be either voltage- or ligand-gated. Ions can
ow only passively with the concentration gradient
through an ion channel.
Transporters are special proteins in the membrane
-
that can pump molecules and ions (also named ion
pumps) against the concentration gradient at the expense of ATP hydrolysis. Many transporters are attractive drug targets, and others are responsible for
the development of drug resistance.
There are alarge variety of known modes of action
-
for drugs. Some of the most diverse modes of action
are found in anti-infective drugs. Furthermore, tumor
therapeutics exploit diverse, toxic modes of action.
The goal in addressing these modes of action in terms
of atherapy is to nd apathophysiological process
that is unique or is as unique as possible to the disease
to spare healthy tissue from damage.
Drug resistance is an increasingly serious problem
-
and is both an inevitable occurrence associated with
using apharmaceutical therapy, and a consequence
of the misuse of anti-infectives. There are several
mechanisms of resistance development in bacteria
or viruses (i.e., fast genetic mutations), and in cancer
therapy (i.e., aberrant transporter expression). These
mechanisms are not mutually exclusive.
The issue of combination drugs is a controversial
-
topic. Some physicians are against them, and others
are in favor of them, and both sides of the argument
have good reasons. Nonetheless, some drug combinations are justiable and help with compliance, clinical
efcacy, and safety.
Special Literature
M. Olah, R. Curpan, L. Halip, A. Bora, N. Hădărugă, D. Hădărugă,
R. Moldovan, A. Fulias, M. Mractc, T. Oprea. Chemical Infor-
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Bibliography and Further Reading
General Literature
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The journals Trends in Pharmacological Sciences, Chemistry & Biology,
Nature Reviews Drug Discovery and Pharmacon contain in each
issue a highly topical article about the mode of action of a biologically active substance
Inhibitors of Hydrolases with
an Acyl–Enzyme Intermediate
Contents
23.1 Serine-Dependent Hydrolases – 372
23.2 Structure and Function of Serine Proteases – 372
23.3 The S
Determines Specicity – 374
23.4 Seeking Small-Molecule Thrombin Inhibitors – 376
23.5 Design of Orally Available Low Molecular
Weight Elastase Inhibitors – 384
23.6 Serine Protease Inhibitors: Thrombin Was
Just the Starting Point – 385
23.7 Serine, aFavored Nucleophile in Degrading Enzymes – 390
23.8 Triads in All Variations: Threonine as aNucleophile – 394
23.9 Cysteine Proteases: Sulfur, the Big Brother of
Oxygen as aNucleophile in the Triad – 396
23.10 Synopsis – 400
Bibliography and Further Reading – 400
Pocket of Serine Proteases
1
© The Author(s), under exclusive license to Springer-Verlag GmbH, DE, part of Springer Nature 2024
G. Klebe, Drug Design, https://doi.org/10.1007/978-3-662-68998-1_23
Chapter • Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate
23
Peptidases and esterases are hydrolytic enzymes, and
2–3% of all gene products can be assigned to this group of
enzymes. They, therefore, represent an important group
of target proteins for the development of new drugs and
are of particular importance for structure-based drug
design. In recent years, many of the known human or
pathogen-derived peptidases have been tested as potential targets for drug therapy.
The function of these enzymes is to cleave peptide or
ester bonds, which requires anucleophile to attack the
carbonyl group of the amide or ester bond to be cleaved.
Alarge number of proteins use the OH or SH groups of
aserine, threonine, or cysteine for this purpose. In the
following chapters, we will see other hydrolyzing enzymes
that use adifferent mechanism. During the cleavage reaction of the hydrolases discussed in this chapter, atemporary covalent bond is formed between the substrate and
the enzyme. This intermediate, called the acyl enzyme
form, occurs with serine, threonine, and cysteine proteases, but lipases, esterases, transpeptidases, and β-lacta-
mases also use this reaction mechanism. The design of
inhibitors for these enzymes that act via an acyl enzyme
intermediate will be discussed. The following two chapters will discuss peptidases that use awater molecule for
the primary attack on the peptide bond to be hydrolyzed:
the aspartic and the metallopeptidases. Depending on
whether they cleave the amino acid chain at the N- or
C-terminus or in the center, peptidases are classied as
aminopeptidases, carboxypeptidases, or endopeptidases.
Some of these proteases are relatively unspecic, while
others are highly specic and cleave only very specic
substrates. It is the latter enzymes that are most likely
to yield aselective therapeutic inhibitor with few side
effects. Bacteria and viruses have also produced their own
peptidases, the inhibition of which can be exploited for
chemotherapeutic treatment. Since these proteins are not
endogenous to humans and, therefore, have no function
in humans, their inhibition should lead to therapeutic
success without the risk of severe side effects.
23.1 Serine-Dependent Hydrolases
Serine proteases are the most abundant and best studied
class of peptidases. They are closely related to esterases
and lipases (hydrolases), which hydrolyze ester bonds.
This class of enzymes serves the human body in many
ways. Some serine proteases, such as the digestive enzymes trypsin and chymotrypsin, cleave awide range of
peptides and proteins. Others, such as the coagulation
enzymes thrombin and factorXa, are highly selective
and cleave only very specic substrates. Often, proteases
are expressed in anonactive precursor form, called zy-
mogens. In order to convert them to their active form,
sequence segments of the zymogen polypeptide chain
are often cleaved, which otherwise serve as endogenous
inhibitors of the activated enzyme. Release of the active
form can occur either by autocatalysis (e.g., trypsin) or
by other activating proteases (e.g., the coagulation cascade). Aserine side chain in the active site plays acrucial
role in the catalytic mechanism of serine proteases, esterases, and lipases. It is characterized by an exceptionally
high chemical reactivity. In chymotrypsin, only this serine reacts with diisopropyl uorophosphate (DFP, avery
potent neurotoxin), while 27other serine residues in the
enzyme remain unmodied. Upon chemical conversion
with DFP, the enzyme completely loses its catalytic activity.
23.2 Structure and Function
of Serine Proteases
The digestive enzyme chymotrypsin was the rst serine
protease to have its 3D structure determined by David
Blow in Cambridge, England. The numbering of the
amino acids in chymotrypsin-type serine proteases is
based on the sequence of chymotrypsin. The three-dimensional structures of alarge number of serine proteases are now available, some of which are listed in . Table23.1. The structures show an extraordinary similarity
in the active site, even for proteases that have completely
different folding patterns (Sect. 14.7, comparison of
trypsin/subtilisin). This so-called catalytic triad Ser–His–
Asp is characteristic for serine proteases. In some of these
enzymes, the aspartate can be replaced by aglutamate,
while some transpeptidases and β-lactamases have alysine instead of the histidine in the active site.
Because these three amino acids are far apart in the
sequence, the protein must fold appropriately to bring
the three side chains into close proximity. The catalytic
serine, located at position 195 in the trypsin-like proteases, performs the actual attack on the amide bond to
be cleaved (. Fig.23.1). The oxygen atom of an unactivated hydroxyl group would not be reactive enough
for this step. Its nucleophilicity, which describes its
tendency to attack an electron-poor carbonyl carbon
atom, is enhanced by the adjacent histidine side chain.
The imidazole side chain of this histidine can accept
aproton from the serine hydroxyl group, allowing the
now negatively charged oxygen atom to nucleophilically
attack the partially positively charged carbon atom of
the amide carbonyl group. The neighboring aspartate
group can take up aproton from the imidazole ring of
the histidine and gives it back again. In this way, it compensates for the positive charge intermediately formed
on the histidine residue. To stabilize the transition state
formed during the attack on the carbonyl group, serine
proteases have another characteristic structural motif,
the so-called oxyanion hole. This is asmall pocket next to
the side chain of Ser 195, composed of two main chain
NH groups (. Fig.23.1). In a few cases, the terminal
ab
cd
. • Structure and Function of Serine Proteases
. Table 23.1 Serine proteases with physiological importance
(X=arbitrary amino acid). The 3D structures of all listed
enzymes are known
Enzyme Cleavage site Function or thera-
peutic approach
Trypsin Arg–X, Lys–X Digestive enzyme
Chymotrypsin Tyr–X, Phe–X,
Trp–X
Elastase Val–X Tissue degradation
Thrombin Arg–Gly Blood coagulation
Factor Xa Arg–Ile, Arg–Gly Blood coagulation
Factor VIIa Arg–Ile Blood coagulation
Tryptase Arg–X Asthma
Matriptase Arg–X Oncology
Urokinase Arg–X Oncology
DPPIV Ala–X, Pro–X Diabetes
Furin Arg–X Viral infection
Digestive enzyme
amide groups of asparagine or glutamine can also perform this function. The function of the oxyanion hole
is to stabilize the negative charge formed on the tetrahedral transition state and to distort the geometry of
the attacked carbonyl carbon atom from atrigonal-planar to atetrahedral conguration. The formed transi-
tion state collapses with the release of the C-terminal
cleavage product, which carries afree amino group at
its terminus. The N-terminal cleavage product remains
covalently bound to the protease to form an acyl enzyme
intermediate. In asubsequent step, nucleophilic attack by
awater molecule again leads to atetrahedral transition
state. This tetrahedral transition state nally collapses,
releasing the N-terminal cleavage product. The catalytic
enzyme is then ready for the next transformation.
What happens when the amino acids serine, histidine,
and aspartic acid of the catalytic triad of a serine protease are individually or collectively replaced by amino
acids without similar functional groups? In 1988, Paul
Carter and James Wells at Genentech produced various mutants of the bacterial serine protease subtilisin
(Sect.14.8). Replacing the catalytic serine or histidine
with alanine reduces catalytic activity by more than six
orders of magnitude. Surprisingly, exchange of aspartic
acid, whose only function is to exchange aproton with
histidine, reduced catalytic activity by more than four
orders of magnitude. The combined exchange of several
amino acids of the catalytic triad did not result in any
further reduction in catalytic activity. The triple alanine
mutant, in which the catalytic triad is completely re-
. Fig. 23.1 Catalytic mechanism of serine proteases. aThe peptide
substrate binds to the enzyme in specic pockets on either side of the
cleavage site. b The oxygen atom of the serine side chain performs
anucleophilic attack. This is facilitated by the adjacent histidine side
chain, which, supported by an aspartate residue, accepts a proton
from the hydroxyl group. cThe transition state collapses to form an
acyl enzyme intermediate. dThis intermediate is hydrolyzed by the
attack of awater molecule to release the N-terminal cleavage product