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3
Chapter • Classical Drug Research
antagonist was to no avail for years. The American management in Philadelphia became impatient and wanted
to stop the program. The rst promising result came just
in the nick of time. Because all lipophilic analogues were
ineffective, the earlier more polar compounds that had
already been investigated were reinvestigated. Acompound that had already been synthesized in 1928 and
determined to be ineffective, Nα-guanylhistamine 3.54
(. Fig.3.11), now appeared to be aweak antagonist.
The effect had been overlooked because 3.54 is actually
a partial agonist and, therefore, shows aweak histamine-like effect. Within afew days the rst lead structure,
S-(2-imidazoyl-4-yl-ethyl)isothiourea 3.55, with interest-
ing activity was identied.
The extension of the side chains of both of these
compounds delivered partial agonists, the antagonistic
effects of which were too weak. It was only in 1972 after
they abandoned the hypothesis that the basic nitrogen
in the side chain was necessary for activity that they,
after chain elongation and an N-methyl substitution of
the thiourea, arrived at the rst clinically useful H2 antagonist burimamide 3.56. Human trials conrmed the
efcacy, but the bioavailability was poor. The next milestone was achieved with the development of metiamide
3.57 (. Fig.3.11), which is 5- to 10-times more potent
than burimamide and clinically demonstrated the desired
ulcer-healing effect. In some patients, however, granulocytopenia occurred, which is adangerous suppression of
white blood cells and cannot be tolerated.
The medical need was great. It was not foreseeable
whether the observed effect was aresult of H2 antagonism. We have the company to thank for taking on the
risk of further research. The sulfur atom of the thiourea
was suspect. An isosteric exchange for an oxygen atom
delivered aless-potent urea analogue. Exchange for an
═NH group led back to guanidine, which was strongly
basic, but apotent antagonist nonetheless. Substitution
of the imino group for an NO2 or aCN group led to
less-basic analogues, the antagonistic potency of which
was comparable to metiamide. The somewhat more active of the two analogues, cimetidine 3.58 (. Fig.3.11),
was clinically tested. In November 1976 and in August
1977, it was introduced in England and the USA, respectively. By 1979, it was available in over 100 countries.
Shortly thereafter in 1983, cimetidine (Tagamet®) became the most-prescribed drug in many countries, and
its sales reached about US$1billion.
Such asuccessful drug makes other companies restless. There are many cases in the history of pharmaceutical research in which amajor new concept was adapted
by developments in other companies. Other examples of
this are the structurally entirely different calcium channel
blockers verapamil and nifedipine (Sects.2.6 and 30.4)
and the angiotensin-converting enzyme inhibitors captopril and enalapril (Sect.25.4).
. Fig. 3.11 Nα-Guanylhistamine 3.54 and S-(2-imidazolyl-4-yl-eth-
yl)isothiourea 3.55 served as lead structures for H2-type antihistamines. The rst clinically tested H2 antagonists, burimamide 3.56 and
metiamide 3.57, were unsuitable for treatment. Only the development
of cimetidine 3.58 led to abreakthrough and an exceedingly successful therapy
The same happened in the development of the H2 antagonists. Ulcer therapy had been researched since 1960
at Allen and Hansburys, asubsidiary of Glaxo. One of
the rst lead structures 3.59 (. Fig.3.12), an aminotetrazole with about the same potency as burimamide,
was systematically varied without success. Their research
management also wanted to stop the project to concentrate on the anticholinergics. The breakthrough came
upon replacement of the tetrazole ring with afuran. It
was not exactly an obvious idea because the previously
synthesized compounds always had at least one nitrogen
atom in the ring. The –CH2SCH2CH2– chain was taken
over from metiamide 3.57, and adimethylaminomethylene group was added to improve water solubility; the
result was AH18665 3.60 (. Fig.3.12).
The chemists also synthesized a cyanoguanidine
AH18801 3.61 that was comparable to cimetidine 3.58
in terms of potency. The substance’s characteristics
were, however, unsatisfactory: the melting point was
too low. The nitrovinyl analogue 3.62 brought success
in this respect. It was synthesized and was an oil! That
was not seen as aprohibitive problem because it was
redeemingly 10-times more potent than cyanoguanidine
3.61 in the rat. Ranitidine 3.62 (. Fig.3.12) was de-
veloped as adrug and introduced in 1981 as Zantac®
and Sostril®. Compared to cimetidine, ranitidine was
4- to 5-times more efcacious in humans and had the
advantage that it was more selective. In 1987, ranitidine overtook cimetidine. In 1994 with US$ 4billion in
sales, it became the most economically successful drug
in annual sales at that time. Within afew years, Glaxo
was catapulted to the pinnacle of the world rankings of
pharmaceutical corporations. Glaxo used this opportunity. The research of this company and its strategy in
drug development belong to “the nest” in the branch
today. Through mergers and acquisitions with compet-
. • H Antagonists: Ulcer Therapy Without Surgery
itors, Glaxo, “GSK” as it is known today, has become
one of the largest pharmaceutical corporations on the
market.
In the meantime, an antitumor effect in colon, gastric,
and renal cancer has been reported for cimetidine. Apparently it suppresses tumor-mediated interleukin-1-induced selectin activation (Sect.31.3).
It is understandable from the chemical structure that
cimetidine has ahigh afnity for cytochrome P450 enzymes, particularly CYP3A4 (Sect.27.6). As aconsequence, interactions with other drugs that depend on
CYP3A4 for metabolism are common. What was rst
seen as an indispensable imidazole moiety in 3.58 blocks
the catalytic iron center in the P450 enzymes. Ranitidine
3.62 carries afuran ring in the same position and lacks
P450 inhibition. After cimetidine and ranitidine, very few
other drugs have made their way to the market. Nizatidine 3.63 and famotidine 3.64 contain athiazole ring as
aheterocycle (. Fig.3.12). In 3.64, the electron-withdrawing group of the guanidine moiety is replaced by
asulfonamide group.
It is true even for the H2 blockers that good drugs are
replaced by better ones. After being prompted to acid
stimulation, the cells use an H+/K+-ATPase active enzyme to pump protons out of the cell in exchange for
potassium at the cost of energy. If “the faucet is turned
off” at this step, not only the histamine-induced acid
production, but also the acetylcholine- and gastrin-mediated acid production is stopped. Omeprazole 3.65 is
aprodrug that has been developed, which, upon rearrangement, acts as an irreversible inhibitor of this proton
pump (Sect.30.9). The effect of omeprazole therefore
lasts longer, and the reduction in acid secretion is stronger than with H2 antagonists. Gastric and duodenal ulcers heal more quickly and reliably. These substances also
hit it big. At the end of the twentieth century, Losec®,
Antra® (both from Astra), and Prilosec® (Merck & Co.,
USA) had combined global sales of over US$ 6billion
despite the fact that they were introduced to the market
much later than ranitidine. The enantiomerically pure
form esomeprazole (Nexium®) even reached US$ 7billion in sales in 2007.
At rst, the use of an enantiomerically pure substance seemed questionable, as the stereogenic center
on the sulfur atom is lost during chemical activation
(Sect.9.5, . Fig.9.13). However, the manufacturing
company was able to demonstrate better bioavailability for the S-stereoisomer, which justies the use of the
enantiomerically pure form. Besides omeprazole, pantoprazole 3.66 in particular was able to achieve high market shares. Together with the follow-up products lansoprazole 3.67 and rabeprazole 3.68, several alternatives
are available that differ in their pKa value and have different rates of activation due to their acid lability. Since
. Fig. 3.12 The lead structures
3.59–3.61 were steps on the way
to ratinidine 3.62, which in the
1980s was the economically most
important drug. Nizatidine 3.63
and famotidine 3.64 represent
newer developments. Omeprazole
3.65 is aproton pump inhibitor.
Further compounds (3.66–3.68)
with aslightly different substitution pattern follow the same
mode of action (Sect.30.9)
Chapter • Classical Drug Research
3
activation occurs in an acidic environment, the active ingredient must rst pass through the stomach, protected
in arm tablet, to then be absorbed via the intestine.
The activation required for the effect nally takes place
after transportation into the gastric cells of the stomach
at the low pH prevailing there. In recent years, proton
pump inhibitors that no longer irreversibly bind to the
protein and also do not require acid-catalyzed activation
(Sect.30.9) have been developed.
Initially, there was concern that prolonged use of proton pump inhibitors would reduce the protective function
of the stomach in killing harmful germs by raising the
pH of the stomach. However, acomprehensive study of
17,600 patients with pantoprazole showed that there was
no statistically signicant difference in inammation other
than enteric infections compared to aplacebo group.
That is not even the end of the story. Although in
principle it had been known since 1983, the relevance
of the bacteria Helicobacter pylori for the etiology of
ulcers was rst discussed in 1994 at aconference of the
US National Institutes of Health (NIH). This bacterium
infects alarge portion of the population in childhood.
Frequently, it is spread within afamily; akiss can be
enough to infect someone. It causes gastrointestinal damage in aportion of those infected, which can lead to an
ulcer. In the meantime, it is held responsible not only for
ulcers but also for at least two different forms of gastric
cancer. It survives assault by many antibacterial agents as
well as the acidic milieu of the stomach. It has aurease
that releases ammonia in its immediate vicinity, which in
turn neutralizes the gastric acid.
The drugs of choice to treat such infections are combinations of H2 blockers, proton pump inhibitors, and
antibiotics. H.pylori seems to quickly develop antibiotic
resistance though. At the beginning of 1995, the rst
animal model, amouse with asustained H.pylori infec-
tion, became available. This promoted further research
in this important area. Avaccine is currently under development. Aportion of the vaccinated patients exerted
enough of an immune response to defend against the
bacteria. For practical use, however, its reliability must
be improved. Perhaps in the foreseeable future, we will
have an ulcer therapy that is completely different, for instance, an oral vaccine that delivers life-long protection.
The revolution is in sight: aone-time treatment without
repeated gastroscopy. Patients will be delighted. Others will see this dramatic change in therapy with mixed
emotions.
3.6 Synopsis
Even though the period of classical drug research
-
was strongly governed by trial and error, it has been
exceptionally successful. Many leads were found by
accident or from traditional medicine, though limited
knowledge of pathophysiology or molecular disease
etiology was available. In many cases, their mechanism of action and interaction with atarget protein
were later elucidated.
Acetylsalicylic acid or Aspirin® is one of our oldest
-
but also most prototypical drugs. Originating from
bark extracts and chemically modied to improve taste
and tolerance, it achieves its actual potency and mode
of action by irreversibly inhibiting cyclooxygenase.
Since then two isoforms of cyclooxygenase have been
-
characterized, one is constitutionally present, and the
other is induced in inamed tissue. Acetylsalicylic
acid inhibits both unselectively, giving rise to some
undesirable side effects.
Due to irreversible inhibition of COX in platelets,
-
Aspirin has an inuence on the ratio of synthesized
thromboxane and prostacyclin, which decreases the
coagulation tendency of blood. As aconsequence,
Aspirin is recommended as “preventive medicine” to
protect against thrombosis or to reduce mortality of
heart attack.
Malaria is awidespread tropical/subtropical disease
-
transmitted by the anopheles mosquito and caused
by the plasmodium parasite accessing erythrocytes in
humans. The disease had been nearly eradicated by
ghting the mosquito with the insecticide DDT. One
of the oldest active substances to use against the parasite is quinine, which is isolated from cinchona bark.
After stopping DDT spraying for the mosquitos, ma-
-
laria raged again. Increasing resistance of the parasite
to known drugs occurred, and the development of
new chemotherapeutics for malaria has been arollercoaster ride of promising compounds and the development of resistant parasites.
Morphine, isolated from poppies and used as the un-
-
changed natural product, is apotent analgesic. When
administered correctly, the risk of addiction is low. Its
complex structure of ve fused rings has been simplied and cut into pieces to give more-easily accessible
analogues with higher selectivity.
Cocaine, which is the active ingredient in coca leaves,
-
is one of our oldest drugs. Upon replacement of dopamine from its transporter in the synaptic gap, its
euphoric effect is achieved. The cocaine structure
served as alead structure for the development of anesthetics.
Ulcer therapy went through several phases of drug
-
development, leading to active substances with increasingly efcient modes of action to reduce production of gastric acid.
Starting with antacids and rather unspecic anticho-
-
linergics, selective H2 antagonists were real breakthroughs in the pure pharmaceutical treatment of
ulcers. They act upon the H2 receptor, amember of
G-protein-coupled receptors (GPCRs). Aprotein that
pumps protons for acid release is stimulated through
Bibliography and Further Reading
these receptors. Proton pump inhibitors such as ome-
prazole directly block the function of the proton-se-
creting H+/K+-ATPase that builds up the acidic milieu.
The bacterium Helicobacter pylori causes gastrointes-
-
tinal damage leading to ulcers. It can be eradicated
by combining aproton pump inhibitor with an anti-
biotic. Avaccine against the bacterium could deliver
life-long protection.
Bibliography and Further Reading
General Literature
G. Ehrhart and H. Ruschig, Arzneimittel. Entwicklung, Wirkung,
Darstellung, 2nd ed., Verlag Chemie GmbH, Weinheim, 1972
A. Burger, A Guide to the Chemical Basis of Drug Design, John Wiley
& Sons, New York, 1983
E. Verg, Meilensteine. 125 Jahre Bayer, 1863–1988, Bayer AG, 1988
W. Sneader, Drug Prototypes and their Exploitation, John Wiley &
Sons, Chichester, 1996
J. Ryan, A. Newman and M. Jacobs, Editors, The Pharmaceutical Cen-
tury. Ten Decades of Drug Discovery, Supplement to ACS Publica-
tions, American Chemical Society, Washington, 2000
W. Sneader, Drug Discovery. A History. John Wiley & Sons, Chich-
ester, 2005
A. Giannis, Naturstoffe im Dienst der Medizin - Von der Tragödie zur
Therapie, Springer-Verlag GmbH, (2023)
W. R. Kelce etal., Persistent DDT Metabolite p,p′-DDE is a Potent
Androgen Receptor Antagonist, Nature 375, 581–585 (1995)
K.-L. Täschner and W. Richtberg, Koka und Kokain. Konsum und
Wirkung, 2.Auage, Deutscher Ärzte-Verlag, Köln, 1988
H. Kubas and H. Stark, Medizinische Chemie von Histamin-H2-Rezep-
torantagonisten, Pharm. u. Z. 36, 24–32 (2007)
University of Oxford: R21/Matrix-M™ malaria vaccine developed
by University of Oxford receives regulatory clearance for use in
Ghana. https://www.ox.ac.uk/news/2023-04-13-r21matrix-m-ma-
laria-vaccine-developed-university-oxford-receives-regulatory
(Last accessed Nov. 29, 2024)
Special Literature
Aspirin – eine unendliche Geschichte, Research. Das Bayer-For-
schungsmagazin, Heft 6, pp 4–21 (1992)
W.-D. Müller-Jahnke and C. Friedrich, Ein Siegeszug mit Hindernissen
– 110 Jahre „Megastar“ Aspirin, Gesch. d. Pharm. 62, 1–11 (2010)
C. Patrono, Aspirin and Human Platelets: From Clinical Trials to
Acetylation of Cyclooxygenase and Back, Trends Pharm. Sci. 10,
453–458 (1989)
B. Battistini, R. Botting and Y. S. Bakhle, COX-1 and COX-2: Toward
the Development of More Selective NSAIDs, Drug News & Per-
spectives 7, 501–512 (1994)
A. Jull etal., Low dose aspirin as adjuvant treatment for venous leg ul-
ceration: pragmatic, randomised, double blind, placebo controlled
trial (Aspirin4VLU), BMJ 359, j5157 (2017)
T.W. Wakeeld etal. An Aspirin a Day to Keep the Clots Away. Can
Aspirin Prevent Recurrent Thrombosis in Extended Treatment for
Venous Thromboembolism? Circulation 130, 1031–1033 (2014)
J.J. McNeil etal. Effect of Aspirin on Cardiovascular Events and Bleed-
ing in the Healthy Elderly, N. Engl. J. Med. 379,1509–1518 (2018)
P.M. Rothwell, M. Wilson, C.E. Elwin, B. Norrving, A. Algra, C.P.
Warlow, T.W. Meade. Long-term effect of aspirin on colorectal
cancer incidence and mortality: 20-year follow-up of ve ran-
domised trials, Lancet 376, 1741–1750 (2010)
R. H. Schirmer and K. Becker, Malaria – Geschichte und Geschichten,
Futura 4, 15–21 (1993)
J. Wiesner, R. Ortmann, H. Jomaa and M. Schlitzer, New Antimalarial
Drugs. Angew. Chem., Int. Ed. Engl. 42, 5274–5293 (2003)
M. Schlitzer, Malaria Chemotherapeutics Part I: History of Antimalar-
ial Drug Development, Currently Used Therapeutics, and Drugs in
Clinical Development, ChemMedChem 2, 944–986 (2007)
H. M. Ismaila etal., Artemisinin activity-based probes identify multiple
molecular targets within the asexual stage of the malaria parasites
Plasmodium falciparum 3D7, Proc. Nat. Acad. Sci, USA, 113,
2080–2085 (2016)
Protein–Ligand Interactions as
the Basis for Drug Action
Contents
4.1 The Lock-and-Key Principle – 41
4.2 The Essential Role of the Membrane – 42
4.3 The Binding Constant
of Protein–Ligand Interactions – 43
4.4 Important Types of Protein–Ligand Interactions – 45
4.5 The Strength of Protein–Ligand Interactions – 48
4.6 Blame It All on Water! – 49
4.7 Thermodynamic Contributions to the Formation of
Protein–Ligand Complexes – 50
4.8 What Is the Contribution of aHydrogen Bond to the
Strength of Protein–Ligand Interactions? – 52
4.9 The Strength of Hydrophobic Protein–
Ligand Interactions – 57
4.10 Binding and Mobility: Compensation
of Enthalpy and Entropy – 58
4.11 Lessons for Drug Design – 62
4.12 Synopsis – 63
K
Describes the Strength
i
Bibliography and Further Reading – 64
© 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_4
Chapter • Protein–Ligand Interactions as the Basis for Drug Action
4
The following question must be answered before adrug
can be specically designed: How do drugs act in general? For example: How does Aspirin® relieve aheadache? Why do β-blockers lower blood pressure? Where
does acalcium channel blocker act? How does cocaine
work? How do sulfonamides prevent the growth of bacterial pathogens? Adrug must bind to aspecic target
molecule in the body to exert its pharmacological action. This is usually aprotein, but nucleic acids in the
form of RNA and DNA can also be targets for active
compounds. An important requirement for binding is
that the drug has the correct size and shape to optimally
t into acavity on the surface of the protein, abinding
pocket. In addition, the surface properties of the ligand
and the protein must be complementary so that specic
interactions can be formed. In 1894, Emil Fischer compared the exact t of asubstrate to the catalytic center
of an enzyme to the picture of alock and key. In 1913,
Paul Ehrlich formulated the Corpora non agunt nisi x-
ata, which literally means “bodies do not act unless they
are bound.” His point was that drugs designed to kill
bacteria or parasites must be “xed,” that is, bound by
certain structures. Both concepts are the starting point
for rational drug discovery. In abroad sense, they are still
valid today. After ingestion, adrug must reach its target
tissue where it interacts with abiological target macro-
molecule. Specic drugs must have sufcient afnity for
the binding site of this macromolecule. Only in this way
can they achieve the required selectivity and develop the
desired biological effect, ideally without signicant side
effects.
The most important terms related to the modes of
action of drugs are briey dened in . Table4.1. These
terms are described in detail in Chaps.23–32 using multiple examples of target structures. Drugs often act as
inhibitors of enzymes or as agonists or antagonists on
receptors. Enzyme inhibitors and receptor antagonists
occupy abinding site (often named orthosteric site) and
prevent the substrate or endogenous ligand from docking
there. Agonists have an additional property called intrin-
sic activity. As aresult, the receptor takes on athree-dimensional structure that elicits aresponse from adownstream process (for details see Chaps.22, 29 and30).
Although ion channels, pores, and transport systems
are also receptors in the broadest sense, they are considered aseparate group. The term “receptor” is often used
. Table 4.1 Brief denitions of the most important terms
Ter m Denition
Ligand A(usually small) molecule that binds to abiological macromolecule
Enzyme An endogenous biocatalyst that can transform one or more substrates into one or more products
Substrate Aligand that is the starting material for an enzymatic reaction
Inhibitor Aligand that prevents the binding of asubstrate either directly (competitive) or indirectly (allosteric),
reversibly or irreversibly
Receptor Amembrane-bound or soluble protein (or aprotein complex) that initiates an effect after binding an
agonist
Agonist Areceptor ligand that exhibits an intrinsic activity, that is, it causes areceptor response
Antagonist Areceptor ligand that either directly (competitive) or indirectly (allosteric) prevents the binding of an
agonist
Partial agonist Aweak agonist that has ahigh afnity to the binding site, and in this way also acts partly as an antag-
onist
Inverse agonist Aligand that stabilizes the inactive conformation of areceptor or ion channel
Functional antagonist Asubstance that prevents areceptor response by another mode of action
Allosteric effector Aligand that inuences the function of aprotein by causing achange in the 3D structure of the protein
Interface antagonist Alow molecular weight ligand that disturbs or blocks the formation of aprotein–protein interface
Molecular glue Alow molecular weight ligand that stabilizes and “glues” aprotein–protein interface together
Ion channel Apore in aprotein that allows specic ions to ow in and out across the cell membrane along acon-
centration gradient. Opening and closing is affected by binding aligand or by amembrane potential
change
Transporter Aprotein that transports molecules or ions across the cell membrane against the concentration gradient
by consuming energy
Antimetabolite Asubstance that interferes with the biosynthesis of acentral metabolic product either as afalse sub-
strate or as an inhibitor
. • The Lock-and-Key Principle
loosely as ageneral term for any biological macromolecule that interacts with adrug.
Beyond the binding of asmall-molecule ligand, biomolecules often communicate with each other by recognizing and forming large shared surface contacts. It is through
these contacts that the primary attack and entry of viruses,
bacteria, and parasites into the host cell takes place. Many
cells receive asignal via surface receptors when they bind
amacromolecule (Chaps.29–32). Even the rolling behavior of leukocytes in the vasculature is controlled by such
surface receptors. These systems are increasingly being
exploited for drug therapy by using active macromolecular substances known as biologicals or biopharmaceuticals (Chap.32). They are increasingly being introduced as
therapeutics in our pharmaceutical arsenal.
4.1 The Lock-and-Key Principle
In the early 1880s, Emil Fischer studied the cleavage of
glucosides with various enzymes that differed only in the
stereochemistry of the glycosidic carbon atom. He noticed that certain glucosides could only be cleaved by one
group of enzymes. Other glucosides could only be cleaved
by another group of enzymes. He drew the correct conclusions from his observations and published them in an
article in Berichte der Deutschen Chemischen Gesellschaft
(Reports of the German Chemical Society) in 1894:
“The limited effect of enzymes on the glucosides can also
»
be explained by the assumption that achemical process
can be initiated only by those [enzymes] that have asimi-
lar geometric construction that approximates that of the
molecule [substrates]. To use apicture, Iwant to say that
enzymes and glucosides must t together like alock and
key to be able to exert achemical effect upon each other.
This idea has gained plausibility and value for stereo-
chemistry research after the phenomena was transferred
from the biological to the chemical eld.”
In the same year he rened this picture:
“Apparently here the geometrical construction exerts
»
such alarge inuence on the play of chemical afnities
that the comparison of the two molecules undergoing
an interaction seems to me to be comparable to alock
and key. If the fact that some yeasts can ferment alarger
number of hexoses than others is to be explained, the pic-
ture can be completed by differentiating between master
and special keys.”
Emil Fischer did not pursue this picture any further and
later even complained that it was often quoted out of
context. He was interested in the conguration of sugars,
but not in the conguration of isomeric glucosides. He
expressed arather distanced attitude to purely theoretical
considerations. In 1912, he wrote in aletter, “Imyself
do not take much pleasure in theoretical things.” This
is remarkably modest for aman who exerted such great
inuence with his image of alock and key! Emil Fischer
would certainly have been pleased and proud to see the
results of X-ray structure determinations of protein–ligand complexes, such as retinol (vitaminA) bound to the
retinol-binding protein, responsible for the transport of
this rather hydrophobic molecule (. Fig.4.1).
The binding of ligands to proteins spans awide range
of specicity and this can occur with different selectivities with respect to the bound ligands. Originally, the
terms specicity and selectivity originate from the study
of enzymes. Specicity refers to the ability of an enzyme
to preferentially convert one or afew substances to any
other analogue. Specic ligands therefore require aparticular chemical substructure that enables the enzyme
to catalytically convert them. Selectivity refers to the selective and potent binding of asubstrate that ideally ts
into the binding site on agiven target protein, ltering
out that substrate (or ligand) relative to alarge number
of other candidates. The extent to which high specicity
or high selectivity is required depends strongly on the biological and cellular context in which aprotein performs
its function.
The following examples will illustrate these aspects.
Enzyme binding sites can be highly specic, especially
for chemically related substrates. For example, they discriminate exceedingly between closely related analogues.
In protein biosynthesis, exquisite recognition of distinct
chemical moieties is required, not even the slightest mishap may occur. Friedrich Cramer investigated the mechanism for the recognition of the amino acids valine and
leucine. These amino acids differ in their side chains only
in that amethyl group is replaced by an ethyl group. The
smaller valine residue should t easily into the “lock” for
aleucine, although it may not bind as strongly. Aclear
distinction, which is absolutely necessary for error-free
protein synthesis, can only occur through repeated recognition. This is indeed the case. An energy-intensive,
iterative, and carefully scrutinized verication process
reduces the error quotient to less than 1:200,000. Because of this strict feedback and control process, even
the correct binding partner is sometimes unsuccessful.
Over 80% are rejected as “questionable.” The result is
aprocess with an accuracy of about 1:40,000.
Due to its function as a transport protein, retinol-binding protein does not discriminate between substrates with high selectivity. The extreme precision as
in protein synthesis is apparently not necessary for its
proper function. In addition to the “stretched” retinol
isomer, the “folded” retinol isomer and chemically related
substances also bind to this protein. Also, other proteins
discriminate very little. Examples of less selective proteins include digestive enzymes (Sect.23.3), metabolic en-
zymes (e.g., cytochromes; Sect.27.6), or the glycoprotein
4
Chapter • Protein–Ligand Interactions as the Basis for Drug Action
. Fig. 4.1 Like akey in alock,
vitaminA (retinol, gray carbon
atoms) ts into the binding pocket of its transport protein. The
surface of the ligand is shown
in green, inside blue. The amino
acids of the protein in the direct
vicinity of the binding pocket are
shown (carbon atoms in orange,
oxygen in red, nitrogen in blue).
To improve the clarity, the back
of the binding site and the residues in front of the binding site
have been omitted.
(7 https://sn.pub/ty7wbA)
GP 170, which is responsible for drug resistance of tumor
cells (Sect.30.10). One bacterial transport protein, oligopeptide-binding proteinA, can bind any peptide with
two to ve amino acids with approximately the same afnity; this is an extreme case of “chemical promiscuity.”
Linus Pauling applied the lock-and-key principle
to the transition states of enzymatically catalyzed reac-
tions. Flexible adaptation often occurs during substrate
binding. The transition state of the reaction binds more
strongly to the enzyme than either the substrate or the
product (Sect.22.3) and it is stabilized by the functional
groups of the binding site. The “lock-and-key” principle
has been repeatedly challenged because of the mobility of the ligand in the binding site; however, even with
amodern high-security lock, the pins are still mobile and
play an essential role in the mechanism.
In the 1950s, DanielE. Koshland proposed the the-
ory of “induced t,” which states that the ligand induces
aconformational change in the protein by binding to it.
The theory assumes aspecic effect, such as enzymatic
cleavage of the substrate. This mechanism does not contradict the lock-and-key principle because, as mentioned
earlier, even ahigh-security lock has moving parts. Small,
induced adaptations play an essential role in the ligand–
receptor complex. Even the relocation of entire protein
domains has been observed. The adaptability of aprotein is usually related to its function. Proteins often need
to be exible enough to perform their biological functions and to recognize the right ligands. In the case of
G-protein-coupled receptors, we are learning more and
more about the importance of intrinsic dynamic behavior
for proper functioning (Sect.29.4).
There are two fundamentally different starting
points for the rational design of ligands, which differ in
the information content of the system. Either the exact
three-dimensional structure of the binding site is known
or it is unknown. In the rst case, the lock is known and
the key “only” needs to be cut and led to the correct
size (Chap.20). In the other case, the active and inactive analogues represent the matching and mismatching
keys. By comparing the keys and systematically varying
them, better tting keys can be designed (Chap.17). In
the following section, the binding of asmall-molecule
drug (“ligand”) to amacromolecular receptor is examined in more detail. These drug targets can be located
outside the cell, inside the cell, or embedded in the cell
membrane. Therefore, we will briey discuss the structure
and function of the cell membrane before focusing on the
protein–ligand interaction.
4.2 The Essential Role of the Membrane
Most biological processes in our body take place inside
cells. These cells are surrounded by amembrane that protects the cellular contents from “leaking.” The membrane
also prevents unwanted xenobiotics from entering the
cell and mediates contacts between cells. Membranes are
also found within the cell, where they form substructures
(called compartments) and separate individual cellular
protein+ligand•protein–ligandcomplex
. • The Binding Constant Ki Describes the Strength of Protein–Ligand Interactions
components. In mammalian cells, the outer membrane
consists of alipid bilayer in which proteins and choles-
terol molecules are embedded (. Fig.4.2). All molecules
can move relatively freely, which is why it is called auid
mosaic membrane.
Such lipid membranes act as barriers for polar substances and as permeable layers for nonpolar molecules.
The importance of membranes for drug transport and
distribution is discussed in detail in Chap.19. Here, only
the important function of the lipid membrane for the
activity of drug molecules is discussed. Membrane-embedded proteins belong to completely different classes.
These include membrane-anchored and membrane-resident enzymes, the large class of G-protein-coupled receptors (Chap.29), ion channels, pores, and transporters
(Chap.30), and surface receptors (Chap.31).
Because of the phosphate and ethanolamine head
groups, the two outer layers of the lipid bilayer are
highly polar. The alkyl chains are on the inside, where
the membrane is nonpolar. Many drugs are also nonpolar and accumulate here in higher concentrations than
in solution. Amphiphilic (soap-like) molecules, i.e., substances that are both nonpolar and polar, arrange themselves in the membrane so that the nonpolar part is on
the inside (. Fig.4.2). This orientation within the membrane is particularly important when the polar group
is apositively charged nitrogen atom, which can form
additional electrostatic interactions with the phosphate
group of the lipids.
Meanwhile, this concept has been experimentally
proven by many independent methods. For many receptors, it is accepted that the ligand binds to asite inside the
protein that is accessible only from the inner layer of the
membrane (e.g., lipases, Sect.23.7; or cyclooxygenases,
Sect.27.9). Therefore, the enrichment and arrangement
of an active molecule in the membrane plays an important role to optimally access the binding site. If, on the
other hand, the molecule assumes an incorrect orientation, its docking to the binding site can be hindered.
4.3 The Binding Constant K
Describes
i
the Strength of Protein–Ligand
Interactions
We would like to quantify the afnity or the strength
of the binding of aligand to its target protein. For this
purpose, the binding of the ligand to its protein is regarded as achemical reaction for which adynamic chemical equilibrium is established after acertain interval of
time. Once this equilibrium is reached, as many ligand
molecules bind to the protein as dissociate from it. Macroscopically, the system no longer appears to change
from the outside; equilibrium is established. For such
asituation, we can formulate alaw of mass action:
Kd=
This constancy of the concentrations of ligand and
protein molecules in the numerator divided by the concentration of the formed complex in the denominator is
expressed as acharacteristic quantity, the so-called equilibrium or binding constant. It is dened as the dissoci-
ation constant Kd (Eq.4.1). Its reciprocal value is called
the association constant Ka. In the case of enzymes, the
so-called inhibition constant Ki is very often determined
in an assay (Sect.7.2) according to Michaelis–Menten
kinetics known from biochemistry. Although not dened
in exactly the same way, the quantities are usually considered and discussed as equivalent in drug design.
Instead of aKi value for the inhibition of an enzyme,
an IC50 value is often given in case of enzyme inhibition.
The IC50 value measures the concentration of an inhibitor
required to reduce the turn-over rate of the considered enzyme reaction by half. Unlike the Ki value, the IC50 value
depends on the concentration of the enzyme and the substrate. Therefore, the IC50 value is also inuenced by the
afnity of the substrate for the enzyme, since substrate
ŒligandŒprotein
Œligand–proteincomplex
= 1=K
a
(4.1)
. Fig. 4.2 Membranes from mammalian cells are constructed from
alipid double layer (“bilayer”), in which proteins (yellow) and indi-
vidual cholesterol molecules (black) are embedded. The individual
lipid molecules (orange) orient their polar groups to the exterior of
the membrane, and their alkyl chains to the interior. Therefore, polar
drugs (light blue) accumulate on the outside of the membrane. Nonpolar drugs (red) are enriched in the interior of the membrane. Amphiphilic drugs (violet) are oriented into the membrane according to their
structure. Despite this, all of the molecules can move relatively freely.
Therefore, this is called a“uid mosaic membrane”
NH
+
ı
−RTln K
d
ı
=RTln K
d
Chapter • Protein–Ligand Interactions as the Basis for Drug Action
4
and inhibitor compete for the same binding site. The IC50
value can be converted to aKi value using the Cheng–Prusoff equation. Experience has shown that IC50 and Ki values are parallel to arst approximation, so that the more
easily determined IC50 value is well suited to characterize
aligand in comparison to other compounds. However,
they should only be used if the data were determined in
the same laboratory and under identical assay conditions.
The afnity thus describes the strength of the interaction between protein and ligand. It is dened by the ratio
of the amount of free to protein-bound ligand. It has the
dimension of aconcentration with the unit mol/L(M).
The smaller its value, the more strongly the ligand binds
to the protein, because then it is mainly the formed complex that is present. If the concentration of the ligand
is signicantly lower than the binding constant, only
avery small proportion of the protein molecules will be
occupied by bound ligand molecules. Abiological effect,
such as the inhibition of an enzyme, can then hardly be
observed. If the free ligand concentration at equilibrium
corresponds exactly to the value of the binding constant,
half of all protein molecules present will be occupied by
ligand molecules.
From chemical thermodynamics, we know an important relationship which connects the binding constant (or
precisely the dissociation constant Kd) with the so-called
Gibbs free energy (∆G). With this expression, the binding constant and the change in Gibbs free energy of the
chemical reaction under consideration, that is the formation of our complex, can be converted into each other
(Eq.4.2). Just as altitude data on ageographic map only
make sense if they are related to areference point such as
sea level, energy data refer to the Gibbs free energy ∆G°
under so-called standard conditions (all binding partners
are present in molar amounts, at T = 298 K and apressure of 1 atm).
(4.2)
In Eq.4.2, Ris the gas constant and Tis the absolute
temperature in Kelvin. If chemical equilibrium has been
reached, ∆G = 0, resulting in Eq.4.3:
It should be noted at this point that in chemistry
a“system-egoistic approach” is applied. This means that
anegative energy value means that the system is releasing
energy, e.g., in an exothermic reaction. Apositive value
means that the system absorbs energy from outside. For
the change in Gibbs free energy, anegative value means
that the reaction runs spontaneously into an equilibrium,
apositive value means that the reaction does not run at all.
But back to thermodynamics. As described above, the
binding constant is related to changes in the Gibbs free
energy. But why do we need such a“free energy” instead
of simply considering aset of energy terms to describe
the energetics leading to the formation of aprotein–ligand complex?
Simply changing afew energy contributions does not
provide acomplete answer to why aparticular process,
such as the formation of aprotein–ligand complex, occurs spontaneously. The following example illustrates this
point. If we take ahot and acold piece of metal and
bring them into contact, everyone knows that heat will
ow from the hot metal to the cold one. The opposite
is not observed, even though the energy content of the
entire system would remain unchanged for this process.
Why does energy spontaneously ow from ahot object
to acold one and not vice versa? This has to do with
the tendency of all-natural processes to distribute energy
evenly. In ahot piece of metal, the metal atoms vibrate
very strongly around their resting positions, which is
why the piece of metal is hot. Some vibrational degrees
of freedom are strongly activated. When the cold metal
block is brought into contact with the hot one, vibrations are transferred. In the end, the metal atoms in both
blocks vibrate similarly about their resting positions,
but on average not as strongly as the atoms in the hot
block had moved before. The total amount of energy
remains the same. However, the energy is now distributed over many more degrees of freedom. Aconstant
energy balance could also be achieved if the hot piece
of metal would get hotter and the cold piece would get
colder by the same amount. But this process does not
(4.3)
The superscript zero to indicate standard conditions is
generally omitted in the specication of ∆G, which is
also the case in the following text. Abinding constant of
Kd = 10−9 M = 1 nM, arespectable value for an active
compound, corresponds to aGibbs free binding energy
of −53.4 kJ/mol at body temperature (to transform to
kcal, divide by 4.184). At 1 µM it is −35.61 kJ/mol, at
1 mM − 17.80 kJ/mol. Achange of Kd by one order of
magnitude means achange of the free energy of binding
by 5.94 kJ/mol (these values have to be compared with
the molecular association energies; . Table4.2).
. Table 4.2 Experimental or quantum mechanically deter-
mined association energies in the gas phase
Dimer Binding energy in kJ/mol
⋯ CH
CH
4
4
C6H6 ⋯ C6H
H2O ⋯ H2O −22
NH3 ⋯ NH
Na+ ⋯ H2O −90
+
Na
⋯ Cl
6
3
⋯ CH3COO
−
−
−2
−10
−18
< −400
< −400