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a
b
c
. • Screening by Using Nuclear Magnetic Resonance
d
e
. Fig. 7.9 In the SAR by NMR method, ligands with weak afnity
to aprotein, in this case stromelysin, are sought from alarge complex mixture. 15N-labeled protein is used and so-called 1H–15NHSQC
spectra are measured. If a ligand such as acetohydroxamic acid7.1
becomes apparent through ashift in the resonance of specic amino acids that protrude into the binding pocket, the binding geometry
can be deduced(a,d). Later the binding site is saturated with these
ligands. Further NMR measurements are carried out to identify ligands for neighboring binding positions. These are revealed by the
shift in the resonances of neighboring amino acids. That is how 4-cya-
amount of time. The magnetic signal is then transferred
from the protein to the bound ligands. Upon dissociation, the changed magnetic properties of the temporarily
bound ligands can be detected spectroscopically because
the relaxation time of the transferred magnetization is
now faster in the uncomplexed state. The solution is measured with and without the magnetized protein. The difference between the spectra is evaluated. Only ligands that
have been bound to the protein in the interim and, thus,
have undergone magnetization transfer will show signals.
The so-called saturation transfer difference (STD) spec-
trum can be used to screen for potentially active ligands
(. Fig.7.8). Many different variations and elaborate ex-
no-4′-hydroxybiphenyl7.2 was discovered(b,d). Achemical coupling
of both hits 7.1 and 7.2 with a –CH2CH2O– linker produced7.3,
which is a nanomolar inhibitor of the protease stromelysin (c, e).
(7 https://sn.pub/zqmC0T)
perimental protocols have been developed for the magnetization transfer principle described above. Even the use
of so-called reporter or spy ligands, which have an easily
measurable NMR signal, can be used. The resonance of
uorine atoms is particularly well suited. The 19Fnucleus
is very sensitive to NMR detection and has awide range
of chemical shifts (see Sect.13.7). In addition, there are
usually only afew uorine atoms present in the systems
under investigation. This requires auorine-containing
reporter ligand that binds to the protein, but the binding
should not be too strong. The ligand should be easily released from the protein by the test ligand. This release is
detectable as achange in the uorine NMR spectrum and,
Chapter • Screening Technologies for Lead Structure Discovery
7
thus, reveals the binding of the test ligand. As explained
in detail in Sect.13.7, the spatial structure of proteins can
be determined by isotopic labeling and the measurement
of mutually coupled NMR spectra (so-called multidimensional spectra). In this way, where atest ligand binds to
aprotein can be accurately determined by evaluating the
specic resonance shifts of the labeled protein. However,
this requires that all resonances be assigned to the atoms
of the protein before the experiment begins. This is usually arather time-consuming task. If the assignment is
not yet available, it is still possible to qualitatively infer
ligand binding from the observation of some signal shifts.
In the best case, it is even possible to see two ligands
binding at the same time, or two different ligands binding at different non-overlapping positions in the binding
pocket. Steven Fesik’s research group at Abbott, USA,
developed this method. It is known as SAR by NMR
(SAR stands for structure–activity relationship) and is
used for lead structure identication and optimization.
This method was used to nd ananomolar inhibitor of
the matrix metalloproteinase stromelysin (Sect. 25.6).
First, apotent head group was sought that could bind to
the zinc ion in the catalytic center of this protease. Such
amolecule, acetohydroxamic acid7.1, was found with
an admittedly weak but specic binding of Kd = 17 mM
(. Fig.7.9). After the discovery of this ligand, the zinc
binding site was saturated with this compound. Further
NMR measurements focused on the search for aligand
capable of lling the adjacent 0 binding pocket. Asmall
library of heteroarylphenyl and biphenyl derivatives was
used for this purpose. 4-Cyano-4′-hydroxybiphenyl7.2
was identied as ahit. The right side of . Fig. 7.9e
shows both ligands in the binding pocket. Evaluation
of the structural data indicated that the hydroxylated
phenyl ring binds near the methyl group of the acetohydroxamic acid. Therefore, linking the fragments was
the next obvious step. An ethylenoxy group was used as
abridge and coupled to the cyanobiphenyl moiety. NMR
spectroscopy conrmed this structural hypothesis and an
inhibitor, 7.3, with an afnity of 25 nM was produced.
7.9 Crystallographic Screening for Small
Molecular Fragments
Crystal structure analysis provides the exact spatial position of amolecule in the binding pocket of aprotein.
Even the geometry of small, very weakly binding molecules is easily recognized. In structures that have aresolution better than 2–2.5 Å (Sect.13.5), water molecules are
usually still visible as discrete density maxima. Often, they
indicate sites in the binding pocket that can be equally
well occupied by polar functional groups of ligands
(. Fig.7.10). In the early 1990s, Dagmar Ringe in the research group of Greg Petzko at Brandeis University, USA,
intentionally exposed protein crystals to solvent molecules
to allow the solvent to diffuse into the crystals. The solvent
molecules can act as probes by populating binding regions
of the protein pockets. As an example, the regions where
isopropanol, acetonitrile, or acetone are encountered in
thermolysin, azinc protease, are shown in . Fig.7.10.
Even phenol, asmall organic molecule, manages to
diffuse into the binding pocket. Phenylsuccinic acid,
alead structure with atypical fragment size, binds to the
zinc protease. Its binding position has been determined by
crystallography. The phenyl ring of this molecule is in the
same position as that explored by phenol. One of the acid
groups of the succinic acid is in the position that was indicated by the carbonyl carbon of acetone. The second acid
group coordinates to the zinc ion and occupies positions
where water molecules resided in the uncomplexed state
(. Fig.7.10). There are many protein–ligand complexes
in which small molecules from the crystallization solution
or cryobuffer have been absorbed. These can be used as
probes to map out abinding pocket. Acreative scientist
will directly exploit their positions for the design of new
drug candidates. From there, it was obvious to use crystal
structure analysis as amethod to screen for the binding of
small molecules or “fragments” (MW < 250 Da).
Crystal structure determination has become increasingly faster in recent years due to data acquisition
at powerful synchrotrons, and data evaluation can be
largely automated. As aresult, crystallographic screening
of fragment collections with up to 1000 test compounds
has become standard practice. However, this requires the
preparation of avery large number of protein crystals
into which the small probe molecules are allowed to diffuse (the so-called “soaking” process, Sect.13.9). Initially,
attempts were made to soak the crystals with a“cocktail”
of several test ligands at the same time in order to speed
up the screening process. However, since this procedure
leads to a large number of problematic artifacts and
hardly interpretable electron density maps (Sect.13.5),
soaking with single compounds has been resumed. Often
very high concentrations of fragments are used, and organic solvents such as dimethyl sulfoxide (DMSO), isopropanol, or dioxane are added to increase the solubility
of the test compounds. Such conditions are often incompatible with the stability of the protein crystals, which
are quite fragile. Under the very harsh conditions, they
lose their diffraction power. It is, therefore, all the more
important to develop strategies that make the protein crys-
tals more robust against the highly concentrated fragment
solutions containing additional organic solvents.
The hits discovered with the initial crystallographic
fragment screening are often only very weak binders in
the millimolar to weak micromolar afnity range. However, they can be optimized very effectively because the
subsequent design process starts from very well-characterized binding modes (Sect.20.7). This is also acrucial
difference compared to hits from, for example, the HTS or
the SPR methods. Whereas the latter methods require hit
. • Tethered Ligands Explore Protein Surfaces
. Fig. 7.10 It was possible to soak small probe molecules (so-called
“fragments”) into crystals of the protease thermolysin. Left Superposition of multiple structures in which water (red spheres), isopropanol (C-atoms are gray), acetone (C-atoms are light blue), acetonitrile
(C-atoms are green), and phenol (C-atoms are violet) had penetrated
the crystals. They describe potential positions for functional groups of
putative ligands. The structure of benzylsuccinic acid, aweakly binding inhibitor of thermolysin, is also shown on the right side. This molecule coordinates with one of its acid groups to the catalytic zinc ion.
Both oxygen atoms of the acid group displace two water molecules
that are present in the noncomplexed structure. The other carboxylate
validation to verify that the binding really interferes with
the mechanism and function of the target, crystallography directly shows where and how the hits have bound.
One approach is to chemically merge two hits found in
different regions of the binding pocket via achemical
linker, analogous to the SAR by NMR method presented
in Sect.7.6. Another, generally more successful, approach
is to chemically extend fragment hits. This involves adding
additional substituents to the original hit molecule based
on the known crystal structure. After such adesign candidate has been synthesized, anew crystal structure is determined. In this way, original hits that serve as seeds can be
grown into enlarged ligands that bind more tightly to the
protein in the binding pocket (Sect.20.7 and . Fig.02,
7 https://sn.pub/4NppHn).
Binding of low afnity fragments by biophysical
methods is often difcult to characterize and reliably
conrm. Different methods often result in conicting hit
rates. Therefore, an alternative strategy using crystal-
lography as the primary screening method has been pro-
posed. Since it reliably determines the binding geometries
of fragments, hits are no longer selected according to
their binding constants, which are very hard to determine
group forms asalt bridge with the neighboring Arg 203. The oxygen of
an acetone molecule was found at almost the same position. The phenyl ring of benzylsuccinic acid was found to occupy almost the same
position as the phenol molecule in the fragment structure. Benzylsuccinic acid can be used as astarting structure for further optimization.
(7 https://sn.pub/7djPkK)
with the necessary condence. Rather, the binding poses
found are evaluated for their relevance to interfering with
protein function and for the possibility of synthetically
extending the placed fragments using well-established
chemistry. If promising candidate fragments emerge according to these criteria, they are chemically enlarged in
acomputer design session. They will then reach asize
that allows reliable assay data to be collected. This approach typically results in high hit rates. If astructure
is then determined from the best follow-up hits of this
second round, single-digit micromolar compounds will
usually be obtained. Since their binding mode is experimentally characterized, they can easily be further optimized in subsequent design cycles.
7.10 Tethered Ligands Explore Protein
Surfaces
Ligands typically bind with very low afnity to at pockets that are open to the surrounding solvent. Therefore, it
is extremely difcult to detect their binding or even to obtain acrystal structure with aligand bound to such asite.
7
Chapter • Screening Technologies for Lead Structure Discovery
. Fig. 7.11 The thiol group of the exposed cysteine is used as an anchor group for the formation of disulde bonds with ligand candidates
from acompound library. There, suitable ligands react that are also able to interact with the surface region in the vicinity of the cysteine thiol.
Acrystal structure was determined from just such acovalently linked complex (. Fig.7.13). After optimization of the initially discovered hit,
the disulde anchor can be discarded and anoncovalent inhibitor can be developed
. Fig. 7.12 From alibrary of 1200 disuldes, the compounds on the side 7.4–7.7 (left) proved to be binders although structurally similar de-
rivatives 7.8–7.11 (right) were synthesized but did not bind to the protein
James Wells and his colleagues at Sunesis in San Francisco, USA, came up with the idea of attaching ligands to
the surface of proteins in order to record their binding to
such pockets. Chemically, this means reacting with the exposed thiol group of acysteine residue on the protein sur-
face. Such acysteine must be present in the native protein
or can be introduced by mutagenesis (Sect.12.2). Under
appropriate reaction conditions, the ligand is anchored by
adisulde bond formed through the thiol group of the
exposed cysteine (. Fig.7.11). Only those test candidates
from the library that are able to interact with the surface
and position themselves with ageometry near the cysteine
thiol group that allows them to react with the sulfur atom
will be bound. In essence, they explore the surrounding
region, react with the cysteine, and remain coupled to the
surface through the disulde bridge. Successfully formed
complexes are then detected by mass spectrometry. James
Wells and Robert Strout chose thymidylate synthase as
their rst target. This enzyme plays an important role in
the de novo synthesis of thymidine, an essential building
block of DNA. Cells with ahigh division rate need this
building block, and thus inhibition of this enzyme could
be apotent anti-infective or antitumor agent (Sect.27.2).
Thymidylate synthase has acysteine residue at position 146, near the catalytic site. From alibrary of 1200
disuldes, compounds 7.4–7.7 proved to be binders,
whereas the very similar derivatives 7.8–7.11 were not
selected (. Fig.7.12). Accordingly, the phenylsulfonamide together with the proline moiety seemed to be
essential for binding. Next, the disulde anchor was
removed, and the binding constant for N-tosyl-d-pro-
line 7.12 was measured to be 1.1 mM (. Fig. 7.13).
. • Tethered Ligands Explore Protein Surfaces
. Fig. 7.13 The millimolar N-tosyl-D-proline 7.12 was optimized in two steps to the nanomolar inhibitor 7.15 by transferring the side chain
from the natural cofactor methylenetetrahydrofolic acid 7.13
. Fig. 7.14 Superpositioning of crystal structures of the enzyme
thymidylate synthase with two tethered ligands, one bound to Cys 143
(C-atoms of ligand7.4 are green) and the other to Cys 146 (C-atoms
of ligand7.4 are violet), both of which are N-tosyl-d-proline derivatives and which are covalently anchored through an S–S bridge. Upon
cleavage of the disulde anchor, the free N-tosyl-d-proline (C-atoms
are gray, 7.12) proved to be aligand with an afnity of 1.1 mM. Its
To further test the concept, Cys 146 was replaced with
aserine residue (. Fig.7.14). Since no binding could be
detected with this mutant, the neighboring His 147 was
mutated to cysteine. However, this mutant was unable to
sh out the N-tosylprolyl residue. In contrast, the mu-
binding geometry is very similar to both of the covalently anchored
derivatives. (7 https://sn.pub/mhW8EO)
tant at position 143 was successful (. Fig.7.14). Here,
aleucine is replaced by acysteine. The subsequently
determined crystal structures showed that the N-tosyl-
prolyl moiety is accommodated almost identically in
the two complexes with the covalent disulde anchor,
Chapter • Screening Technologies for Lead Structure Discovery
7
as it is bound in the complex without the S–S anchor
(. Fig.7.14). This is convincing evidence that the covalent attachment does not enforce the binding geometry.
Rather, the method allows small, initially weakly binding ligands to be shed out of alarge library. Ananomolar inhibitor 7.15 was developed from the initial
millimolar hit 7.12 by transferring the side chain of the
natural cofactor methylenetetrahydrofolic acid 7.13 in
two steps (. Fig.7.13).
The tethering method can be applied quite generally. It has been particularly successful in the search for
ligands that disrupt the formation of protein–protein
surface contacts (Sect.29.8). Amajor advantage of this
technique is that it does not require the development of
an additional biochemical binding assay. Weakly binding
ligands are covalently “tethered” and cannot be washed
away as in the case of simple complex formation. In addition, the covalently bound chemical probes allow the
adaptive capacity of the surface region to be explored.
7.11 Synopsis
Large substance libraries are screened for biological
-
effects to lter out active molecules and assess their
value for agiven indication.
Three phases are distinguished: abroad automatic in-
-
troductory screening for hits, amore detailed screen-
ing of chemical analogues around ahit to establish
arst structure–activity relationship, and lead opti-
mization to nd candidates for clinical testing.
A prerequisite for high-throughput screening was
-
the development of in vitro test systems using pure
proteins produced by gene technology along with the
entire arsenal of biochemical methods in the test tube
so that the function of single-gene products can be
recorded.
As adisadvantage, high-throughput screening does
-
not assess the entire effect spectrum and ignores ef-
fects such as transport, distribution, metabolism, and
excretion.
Screening libraries are frequently assembled of mole-
-
cules from other drug development projects; as such,
they are rather inefcient with regard to their molec-
ular size and their modest screening hit activity in the
micromolar range. Small substances with high ligand
efciency and sufcient space for structural optimiza-
tion are particularly promising.
Enzymatic function and its inhibition can be recorded
-
by the production of chromophoric reaction products.
Radioactively labeled compounds or enzyme-linked
-
immunosorbent assays are versatile techniques to re-
cord protein function on the molecular level.
Progress in assay miniaturization calls for sophisti-
-
cated robotic systems, ever-improving sensitivity of
the read-out, including uorescence measuring tech-
niques, and reliable logistics to handle the enormous
data ow.
Aggregate formation of hydrophobic test compounds
-
can exert signicant inuence on the assay read-out
or even cause false-positive or -negative hits.
Testing on cell-based assays is performed to study
-
changes in cellular- or organism-related function
beyond pure binding of atest compound to agiven
protein target.
Primary animal testing in vertebrates has been abol-
-
ished today for ethical reasons, but it is being increasingly replaced by whole-animal screening using nematodes as the simplest multicellular organism to record
synergistic and side effects.
As acomplementary and alternative method, virtual
-
computer screening has been developed to screen
large compound libraries by docking ligand candidates into the known spatial structure of atarget protein.
Binding events are recorded by biophysical methods
-
such as surface plasmon resonance, thermal stability
shifting, mass spectrometry, uorescence methods,
microscale thermophoresis, or microcalorimetry.
They are used to detect ligands as potential binders.
NMR spectroscopy can be used to detect ligand bind-
-
ing by magnetization transfer. Multiple binders can
be chemically linked to more strongly binding ligands
according to the SAR by NMR technique.
Exposure of small molecular probes and fragments to
-
protein crystals allows for structural characterization
of the binding modes of weakly binding fragments as
aversatile starting point to lead optimization.
Small-molecule fragments tethered to a protein
-
through covalent attachment to the exposed thiol
group of acysteine residue allow the exploration of
the binding properties of at, solvent-exposed surface
depressions and serve as astarting point to develop
antagonists to perturb the protein–protein interface
in complex formation.
Bibliography and Further Reading
General Literature
M.T.S. Stubbs and G. Klebe, in Die Pharmaindustrie, Eds. D. Fischer,
J. Breitenbach, Spektrum Akademischer Verlag, Heidelberg, Berlin
(2005)
L.M. Mayr and P. Fürst, The Future of High-Throughput Screening,
J. Biomol. Screening, 13, 443–448 (2008)
M. Vogtherr and K. Fiebig, NMR-Based Screening Methods for Lead
Discovery pp. 183–202, in Modern Methods of Drug Discovery, Ed.
A. Hillisch und R. Hilgenfeld, Birkhäusen Verlag (2003) ISBN:
376436081X
G. Klebe, Virtual Ligand Screening: Strategies, Perspectives and Lim-
itations, Drug Discov. Today, 11, 580–592 (2006)
T. L. Blundell, H. Jhoti and C. Abell, High-Throughput Crystallogra-
phy for Lead Discovery in Drug Design, Nat. Rev. Drug Discov.,
1, 45–54 (2002)
Bibliography and Further Reading
P. J. Hajduk and J. Greer, A Decade of Fragment-based Drug Design:
Strategic Advances and Lessons Learned. Nat. Rev. Drug Discov.,
6, 211–219 (2007)
M. M. Siegel, Early Discovery Drug Screening Using Mass Spectrome-
try, Current Topics in Medicinal Chemistry, 2, 13–33 (2002)
A. K. Jones, S. D. Buckingham and D. B. Sattelle, Chemistry-to-Gene
Screens in Caenorhabitis Elegans, Nat. Rev. Drug Discov., 4, 321–
330 (2005)
S. Löfås, Optimizing the Hit-to-Lead Process Using SPR Analysis. As-
say Drug Dev. Technol., 2, 407–415 (2004)
W. Jahnke and D. A. Erlanson, Fragment-based Approaches in Drug
Discovery, Vol. 34 in Methods and Principles in Medicinal Chem-
istry, R. Mannhold, H. Kubinyi und G. Folkers, Eds., Wiley-VCH,
Weinheim (2006)
D. A. Erlanson, S. W. Fesik, R. E. Hubbard, W. Jahnke and H. Jhoti,
Twenty years on: the impact of fragments on drug discovery, Nat.
Rev. Drug Discov., 15, 605–619 (2016)
Special Literature
D. Cubrilovic, A. Biela, F. Sielaff, T. Steinmetzer, G. Klebe, R. Zenobi,
Quantifying protein–ligand binding constants using electrospray
ionization mass spectrometry: A systematic binding afnity study
of a series of hydrophobically modied trypsin inhibitors, J. Am.
Soc. Mass. Spectr., 10, 1768–77 (2012)
L. Piersimoni, P. L. Kastritis, C. Arlt, A. Sinz, Cross-Linking Mass
Spectrometry for Investigating Protein Conformations and Pro-
tein−Protein Interactions − A Method for All Seasons, Chem.
Rev., 122, 7500–7531 (2022)
P. Dumas etal., Extending ITC to Kinetics with kinITC, Methods in
Enzymology, Vol. 567, Chap. 7, A. Feig Ed. (2016)
M. Jerabek-Willemsen, T. Andre, R. Wanner, H. M. Roth, S. Duhr, P.
Baaske, D. Breisrecher, MicroScale Thermophoresis: Interaction
analysis and beyond. J. Mol. Struct., 1007, 101–113 (2014)
A. J. Gupta, S. Duhr, and P. Baaske, Microscale Thermophoresis
(MST). In Encyclopedia of Biophysics, G. Roberts and A. Watts,
Eds. (Berlin, Heidelberg: Springer Berlin Heidelberg), pp. 1–5
(2018).
J. Niemax, R. Strasser, P. Hampel, U. Rant, Analyse von Biomolekülen
mit aktiv bewegten Nano-Oberächen, Laborwelt, 11, 21–22 (2010)
P. J. Hajduk, G. Sheppard, D. G. Nettesheim, E. T. Olejniczak, S. B.
Shuker, R. P. Meadows, D.H. Steinman, G. M. Carrera, Jr., P. A.
Marcotte, J. Severin, K. Walter, H. Smith, E. Gubbins, R. Simmer,
T. F. Holzman, D. W. Morgan, S. K. Davidsen, J. B. Summers and
S. W. Fesik, Discovery of Potent Nonpeptide Inhibitors of Stro-
melysin Using SAR by NMR, J. Am. Chem. Soc.,119, 5818–5827
(1997)
C. Mattos and D. Ringe, Locating and characterizing binding sites on
proteins, Nat. Biotechn., 14, 595–599 (1996)
D. A. Erlanson, A. C. Braisted, D. R. Raphael, M. Randal, R. M.
Stroud, E. M. Gordon and J. A. Wells, Site-directed Ligand Dis-
covery, Proc. Natl. Acad. Sci. U.S.A., 97, 9367–9372 (2000)
J. Müller etal., Magnet for the Needle in Haystack: “Crystal Structure
First” Fragment Hits Unlock Active Chemical Matter Using Tar-
geted Exploration of Vast Chemical Spaces, J. Med. Chem., 65,
15663–15678 (2022)
Optimization of Lead
Structures
Contents
8.1 Strategies for Drug Optimization – 116
8.2 Isosteric Replacement of Atoms and Functional Groups – 117
8.3 Systematic Variation of Aromatic
Substituents: The Topliss Trees – 118
8.4 Optimizing the Activity and Selectivity Prole – 118
8.5 From Agonists to Antagonists – 119
8.6 Optimizing Bioavailability and Duration of Action – 120
8.7 Variations of the Spatial Pharmacophore – 121
8.8 Optimizing Anity, Enthalpy, and Entropy of
Binding and Binding Kinetics – 122
8.9 Synopsis – 125
Bibliography and Further Reading – 125
© 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_8
Chapter • Optimization of Lead Structures
8
A lead structure is the starting point on the way to
adrug. The potency, specicity, and duration of effect
must be optimized, and the side effects and toxicity must
be minimized in ausually elaborate, iterative process. Every change in the chemical structure modulates the 3D
structure of the molecule, its physicochemical properties,
and the activity spectrum. The isosteric replacement of
atoms or groups, the introduction of hydrophobic building blocks, the dissection of rings or the restriction of
exible molecular portions into cyclic structures, and the
optimization of the substitution pattern are all possibilities to purposefully modify aputative lead structure.
Creativity and luck are always important prerequisites for success in pharmaceutical research. Nonetheless,
there is atreasure chest of decades of accumulated expe
rience that can be exceedingly supportive to the rational
optimization process. Computer-aided methods can contribute to their full capability in this eld in particular.
Several general considerations and approaches to lead
optimization are presented in the sections of this chapter.
Adiscussion of the structure-based and computer-aided
optimization of lead structures is presented in Chaps.17
and20. Examples for its application to different therapeutic areas are presented in Chaps.21, 23–32.
8.1 Strategies for Drug Optimization
The optimization of active substances follows aprocess
that is best characterized by the words of the philosopher
Sir Karl Popper:
“The truth is objective and absolute. But we can never be
»
sure that we have found it. Our knowledge is always an
assumed knowledge. Our theories are hypotheses. We test
for the truth in that we exclude what is false.” (Objective
Knowledge, 1972)
Accordingly, the optimization of acompound’s potency
follows aworking hypothesis, while an iterative process
of trial and error renes the hypothesis. The accumulated
data on the relationship between chemical structure and
biological activity is used to design new structures. These
are synthesized and tested, and anew working hypothesis
is modied as appropriate. In negative cases, the hypothesis is discarded and anew one is formulated that better
ts the biological data.
The following qualities are distinguished in the structure of the compound:
The actual pharmacophore (Sects.8.7 and17.1) that
-
is responsible for the specic binding and upon which
only limited chemical modication can be carried out,
The additional groups (adhesion groups) that improve
-
afnity and biological activity,
Further groups that do not inuence the binding but
-
rather the lipophilicity of the molecule and with it
the transport and distribution in biological systems
(Chap.19), and
The groups that must be cleaved or modied in the
-
organism to release the actual active form (Chap.9).
The most important steps in the optimization of lead
structures are the systematic changes in the shape and
form, that is, the three-dimensional structure, and/or
the physicochemical properties. Single steps along this
route are
Changes in the lipophilicity and the electronic prop-
-
erties through the introduction or removal of hydro-
-
phobic or hydrophilic groups,
Variations of substituents at aromatic or heteroaro-
-
matic rings,
Introduction or elimination of heteroatoms in chains
-
or rings,
Changes in chain length of aliphatic groups or linkers,
-
Introduction of space-lling substituents to stabilize
-
aparticular conformation,
Changes in the ring size of alicyclic or heterocyclic
-
rings,
Incorporation of exible partial structures in rings,
-
Incorporation of branches or attachments to rings
-
(rigidifying),
Opening of rings,
-
Elimination of chiral centers to simplify astructure,
-
Addition of chiral centers to increase the selectivity,
-
or
Shifts of the thermodynamic binding prole and the
-
drug’s residence time at the target protein.
These processes are usually unidirectional in classical
drug optimization, that is, the optimization takes place
on one position of the molecule at atime, in one single
direction. In the past, such unidirectional optimization
has led to many disappointments because interdependent
inuences of the structural changes were neglected, or
the optimal lipophilicity was exceeded. John Topliss developed ascheme for the variation of aromatic substituents that allows the biological activity to be optimized in
aminimum number of steps (Sect.8.3). The application
of experimental design, simultaneously changing multiple parts of amolecule, and the evaluation of the results
by using quantitative structure–activity relationships
(Chap.18) usually allows fast and effective optimization.
In structure-based and computer-aided optimization, the
3D structure of the target protein and its complexes leads
to directed structural variations of the active substances.
Here again, the aspects of total lipophilicity and metabolism should not be neglected.
. • Isosteric Replacement of Atoms and Functional Groups
8.2 Isosteric Replacement of Atoms
and Functional Groups
Isosteric replacement is the exchange of particular groups
in a molecule for sterically and electronically related
groups. If the biological effect is essentially maintained,
the term bioisosteric replacement (. Fig. 8.1) can be
used. In the simplest case, asingle atom is exchanged, for
instance, aCl (lipophilic, weakly electron withdrawing)
is replaced by aBr (same characteristics as Cl) or methyl
(lipophilic, weakly electron donating), or an –O– (polar, H-bond acceptor) is exchanged for an –NH– (polar,
H-bond donor) or a–CH2– (lipophilic, unable to form
H-bonds) group. Furthermore, bioisosteric replacement
also means the exchange of entire groups. For example,
–COOH, an H-bond acceptor and donor, can be replaced with other groups that have the same or modied
properties, for instance, with the similarly acidic tetrazole. Another example can be found in the exchange of
aphenyl ring for athiophene or afuran building block
(. Fig.8.1). The potential of isosteric replacement is
illustrated in the exchange of all three iodine atoms of
triiodothyronine T3 8.1 for alkyl groups to give 3,5-dimethyl-3′-isopropylthyronine8.2, which in turn retains
impressive afnity and agonistic activity on the thyroid
hormone receptor. In contrast to triiodothyronine, which
is both iodinated and metabolized by adeiodinase, the
alkyl groups of8.2 are no longer metabolically cleavable.
Bioisosteric replacement is one of the most important
strategies in pharmaceutical drug optimization. Nonetheless, surprises sometimes occur. The replacement of
an ester for an amide group in local anesthetics (Sect.3.4)
. Fig. 8.1 A few possibilities for the isosteric replacement of atoms
and/or groups
. Fig.
8.2 Isosteric replacement with retention, loss, and reversal of
the biological activity. All three iodine atoms of the thyroid hormone
thyroxine8.1 can be replaced with alkyl groups and compound8.2 is
still active. In the case of acetylsalicylic acid8.3, the exchange of the
–OCOCH3 for an NHCOCH3 group led to the loss of the acylating
ability and, therefore, anearly complete loss of the biological activity.
The antimetabolite sulfanilamide8.4 (R = SO2NH2) is derived from
p-aminobenzoic acid8.4 (R = COOH), which is acritical intermediate
in the bacterial dihydrofolate synthesis; 8.4 (R = SO2NH2) is the result
of the exchange of acarboxyl group for an isosteric sulfonamide group
expectedly improved the metabolic stability. In the case
of acetylsalicylic acid 8.3 (. Fig. 8.2), this exchange
cannot be made. An analogous exchange of the –COO–
group for a–CONH– group results in acomplete loss
of activity because the amide can no longer acylate the
cyclooxygenase enzyme (Sect.27.9). In the case of p-am-
inobenzoic acid (R = –COOH, . Fig.8.2) the exchange
of acarboxyl group for asulfonamide group gives sulfanilamide8.4 (R = –SO2NH2), which is an antimetabolite
of p-aminobenzoic acid (Sect.2.3).
Alead structure is rarely studied exclusively by one
research group. Other companies adopt successful examples, at the very latest after the economic success of
anew medicine. The goal of this so-called “me-too” re-
search is to modify the competitor’s lead structure to
arrive at patent-free analogues that are more efcacious,
more selective, or better tolerated. It must be accepted
that even this form of competition has led to the therapeutically most valuable compounds in many therapeutic
areas. On the one hand, aplentitude of duplicate work
has been performed, while on the other hand, new analogues with improved properties have been produced and
introduced to therapy which turned out to be successful
in the long run. Penicillins of the third and fourth generation with broad-spectrum activity and metabolic stabil-
β
-blockers with improved selectivity, and many other
ity,