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. • Getting Faster and Faster: More and More Compounds by Using Less and Less Material
. Fig. 7.1
the terminus of a natural protease or esterase substrate. The enzyme
cleaves the p-nitrophenolate or p-nitroanilide, which becomes visible as
ayellow-colored mesomerically stabilized anion (absorption maximum
Ap-nitrophenolate or ap-nitroanilide group is added to
as ametric for optimization need to be considered. LE
refers to ∆G, which is recommended to be measured in
the SI unit kJ/mol. However, values based on the non-SI
unit kcal/mol are more commonly used. Although the
energy scales differ only by the factor of 4.184, care must
be taken to ensure which scale is being referred to. Furthermore, an LE
of 0.3–0.4 (or LE
kcal/mol
kJ/mol
of 1.2–
1.6) is considered apromising value. However, there are
successful drugs in therapy that have an LE that is twice
as high, and others that have an LE that is only half as
high. Therefore, LE should only be used when optimizing
one class of compounds for agiven target.
7.2 Color Change Demonstrates Activity
Important target proteins for drug development are proteases and esterases, which are enzymes that cleave peptide and ester bonds (Chaps.23, 24, 25). How can their
enzymatic activity be visualized? In order to achieve this
goal, synthetic substrates that are very similar to the natural substrate are prepared. For example, they carry apa-
ra-nitroanilide or apara-nitrophenolate group coupled
to the peptide or ester bond to be cleaved (. Fig.7.1).
When the enzyme degrades this substrate, yellow nitrophenolate or nitroanilide is released, and the absorption properties of the produced anion are ameasurable
change. This can be observed spectroscopically. If during
screening, acompound acts as an inhibitor, the enzymatic cleavage of the synthetic substrate will be more or
less suppressed, and the yellow color is minimized. In
this way, the inhibition potency of test substances can
be determined (. Fig.7.1).
Abroad palette of chromophoric reactions that are
suitable for the characterization of enzymatic activity
have been developed. Many enzymes, for example, dehydrogenases, need NAD(P)H as a natural cofactor,
which is subsequently oxidized to NAD(P)+ (Sect.27.1).
at 405 nm). If acompetitive inhibitor is added along with the substrate
to the enzyme, the cleavage reaction rate is suppressed depending on
the binding strength. This is apparent by the more or less strong yellow
color of the solution, which can be quantitatively measured
Because the NAD(P)H starting material, in contrast to
the product, absorbs at 340 nm, the progress of the enzymatic reaction can be monitored at this wavelength.
As avariation, two enzymatic reactions can be coupled
to each other. This route is of interest when the reaction
in the enzyme of interest is difcult to observe, but the
reaction forms aproduct that is asubstrate suitable for
spectroscopic monitoring. In this case, the activity of the
enzyme of interest is recorded based on the conversion of
the product of the previous reaction in the subsequent enzyme reaction. Although absorption spectroscopic assays
are preferred for technical reasons, tests that are based
on the reaction of radiolabeled compounds still play an
important role. The activity of kinases is, for example,
followed by using 32P-labeled adenosine triphosphate.
The terminal phosphate group of the labeled substrate is
transferred to the phosphorylated protein by the kinase
(Sect.26.3). The incorporation rate serves as ameasure of
the kinase activity. Receptor-binding studies are carried
out with aknown radioactively labeled ligand. The assay
investigates to what extent test compounds can displace
the radioactively labeled ligand from the receptor-bind
ing site. This type of test does not necessarily represent
afunctional assay though. Agonistic and antagonistic
binding (Chaps.28 and29) must still be distinguished.
7.3 Getting Faster and Faster: More and
More Compounds by Using Less and
Less Material
Antibodies play an important role in assay development. The enormous specicity of antibody–antigen
interactions can be exploited as ahighly sensitive system (Sect.32.3) for the detection of aspecic molecular
species. In classical immunoassays, either the release of
aradioactively labeled substance is monitored (radioim-
-
Chapter • Screening Technologies for Lead Structure Discovery
7
munoassay, RIA) or an enzymatic reaction is induced (enzyme-linked immunosorbent assay, ELISA). In the latter
case, for example, aspecic antibody is used to nd any
unconverted substrate molecules left over from the enzyme reaction being tested for inhibition. Any antibody
not bound to asubstrate molecule is then removed. Since
the antibody is also linked to another enzyme that can
produce achromogenic compound as reaction product,
the amount of unreacted substrate molecules and, thus,
the inhibition rate of the tested enzyme can be determined. The ELISA technique has amuch wider range of
application, mainly because radioactivity is best avoided
as a measured quantity. Because they recognize only
asingle molecular species, immunoassays are not only
highly specic but also versatile.
Screening techniques are optimized to be automated
and miniaturized. Driven by the desire for higher ca-
pacity, these tests are hardly ever carried out in 96-well
(8 × 12) microtiter plates anymore. The wells of these
plates hold areaction volume of about 0.3 mL. In the
meantime, 384-well (16 × 24) microtiter plates are used
or even 1536-well (32 × 48) plates, the volumes of which
are only afew microliters per well. The aggregation be-
havior of hydrophobic test compounds poses alarge prob-
lem. The aqueous buffer solutions that are used for these
assays can cause these compounds to aggregate. This aggregation creates hydrophobic surfaces on which proteins
can adsorb. This reduces the concentration of free protein, which can give the impression that the protein is well
inhibited. Addition of detergents can reverse this effect.
However, aggregation of test molecules and proteins on
such aggregates, or even on the plastic material of the
assay plates, can dramatically obscure assay results.
By using asophisticated robot system, 100,000 assays
aday can be carried out. This leads to an enormous ood
of data to be evaluated. The reduced test volume has
the advantage that much less material is consumed. Furthermore, the measurements can be carried out quickly.
At the same time, the sample manipulation has become
ever more difcult. One only has to consider the evaporation of such small amounts of solution, the enormously
increasing logistics of comprehending so much data in
parallel, or the reproducibility of the results, and the necessary sensitivity to measure weak signals with certainty
to appreciate the difculty.
In order to improve this last aspect, ever more sensitive detection procedures are used. Fluorescence measur-
ing techniques are particularly sensitive. In the simplest
case, auorescing substrate such as coumarin (Sect.14.6)
is incorporated in the place of para-nitroanilide. The protein–ligand binding can also be followed by uorescence
anisotropy (or polarization). For example, aknown small
molecule ligand for the target biomolecule to be screened
is coupled to auorophore and excited with polarized
light. The emitted uorescence is also polarized. During
the time that the excited molecule can freely diffuse in
solution, the amount of induced polarization decreases.
Because asmall molecule can diffuse much faster than
alarge target biomolecule, the rate by which the polarization signal decreases depends on whether the small
molecule is bound to the large biomolecule or not. In
ascreening assay, the uorophore-labeled ligand is used
as areporter ligand and will be displaced by potential
screening hits. Any difference in polarization decay is recorded and related to the binding event of ahit.
Even better sensitivity can be achieved with so-called
FRET measuring techniques (fluorescence resonance energy transfer). Aresonance energy transfer can occur
between donor and acceptor uorophores of similar absorption if both are separated by no more than 50 Å. For
example, to develop aphosphatase assay, aphosphorylated peptide substrate must be covalently linked to adonor uorophore. This is added to the phosphatase to be
tested, which has also been exposed to an inhibitor. Depending on the inhibitory potency of the test compound,
the activity of the enzyme decreases and less substrate is
cleaved. An antibody that binds to the unused phosphorylated substrate is added. The antibody is additionally
labeled with an acceptor uorophore whose absorption
maximum overlaps with the emission spectrum of the
donor uorophore. If there is still alot of phosphorylated substrate present, e.g., because the compound to
be tested is apotent inhibitor, the spatial proximity between the donor and acceptor uorophores will result in
astrong FRET signal. Thus, the intensity of the FRET
signal can be related to the activity of the tested enzyme.
In the meantime, progress in assay miniaturization allows the detection of single molecules. This is possible by
using uorescence correlation spectroscopy (FCS). Acon-
focal laser microscope irradiates approximately afemtoliter of test solution. If asingle uorophore diffuses
through the volume of interest, it will cause atime-resolved uctuation in the uorescence signal. An exact
analysis of these signals delivers information about the
concentration and diffusion constants. The diffusion velocity, on the other hand, depends on whether the uorescence-marker-labeled substance is bound to aprotein
or not. If the proteins as well as the ligands are tagged
with different markers, the association and dissociation
can even be followed.
7.4 From Binding to Function:
Testing in Entire Cells
The binding of a ligand to a protein does not tell us
much about the associated biological function or the
change in function that is induced. In the case of enzymes that catalyze a chemical transformation, it is
often easier to relate an observed assay inhibition to
a putative function. The correlation is less obvious
with receptors and ion channels (Chaps. 28, 29, 30).
. • Back to Whole-Animal Models: Screening on Nematodes
. Fig. 7.2 Genes are controlled by promoters. Pro-
moter-initiated gene activation leads to the synthesis of
the relevant protein. By using green uorescent protein
(GFP), an easily observed assay can be constructed
based on this principle. For this, the gene promoter
that is activated by agonist binding is coupled to the
GFP gene. Activation of the promoter then delivers
not the original gene product, but rather the GFP. The
presence of GFP is easily observed because of its uorescence upon excitation with ultraviolet light
When biochemical pathways and cell cycle regulation
are considered, it becomes even more complex to assign aparticular function even in case of enzymes. Such
correlations are not so easily reproduced in atest tube.
Therefore, assays must also be developed to study function that allow the response of an entire cell to be measured upon ligand binding. It is possible to culture cells
for many different tissues, which then allows the study
of tissue-specic receptors.
Typically, the activity of ion channels can be investigated by using binding tests or radioactive assays. The
so-called patch–clamp technique allows the inuence of
adrug candidate to be even better characterized. An electrode is placed on the surface of acell and avoltage is
applied, which creates acurrent. In this way, the opening
or closing of individual ion channels can be recorded,
especially when test molecules are added during these
measurements. This method certainly does not have the
capacity of high-throughput techniques. Rather, it is used
to further investigate the function of hits from an initial
prescreening. Fluorescence methods are often used for
this rst step. For example, in the case of calcium channels, the increase in intracellular calcium concentration
can be observed using adye that uoresces sensitively in
the presence of calcium ions.
Other tests employ the coupling to areporter gene.
Receptor stimulation initiates asignaling cascade that,
for some receptors, leads to the transcription of gene
products that are controlled by the relevant promoters
(Sect.28.1). If the sequence of the relevant gene is replaced with that of areporter’s, such as β-galactosidase,
luciferase, or green uorescent protein (GFP), then these
proteins will be produced by the cell instead. This can
subsequently be observed as an easily detectable signal
(. Fig.7.2). As examples, if the produced β-galactosi-
dase cleaves X-gal, ablue dye will be released, luciferase
will develop an ATP-dependent chemiluminescence, and
the green uorescent protein will be detectable because
of its own intrinsic uorescence.
7.5 Back to Whole-Animal Models:
Screening on Nematodes
Primary substance testing on animals, as it was once carried out, is ethically unjustiable today. Furthermore, an
animal model is not predictive for target-oriented optimization. Nevertheless, it does have advantages. The re-
action of an entire organism to asubstance is immediately
transparent, the bioavailability is directly measured, and
side effects as well as synergistic effects are immediately
obvious. Back in 1963, Sydney Brenner recognized the
complexity of molecular biology in that he emphasized
the biochemical control of cellular development. He
proposed that the pinworm (the nematode Caenorhabdi-
tis elegans) would be the simplest multicellular organism
to investigate. This nematode normally lives in soil and
feeds on bacteria. It is also easily culturable in microtiter plates and fed with Escherichia coli bacteria. It is
ahermaphrodite, has ashort lifespan, reproduces itself
within three days, can be conserved in liquid nitrogen, is
transparent, and homologous genes have been found in
humans for 60–80% of its genes. The pinworm genome
has been sequenced, and we now understand how to easily manipulate it. Because it is transparent, any internal changes can be easily observed so that, for instance,
proteins can be tagged with uorescence markers. Its
959 somatic cells form many different organs, including
anervous system with 302 neurons. Can substance testing be carried out in such alife form? The ethical thres-
Chapter • Screening Technologies for Lead Structure Discovery
7
hold may be set lower in this case. But then, how predictive would any tests be? Can such an animal be used
to predict mood changes, depression, or appetite and
its relation to obesity? This is only possible if the causes
of these diseases are known on the molecular level, for
example, adefect caused by an altered serotonin-mediated signaling. In such asituation, the worm can serve
as amodel. Arst step towards the discovery of apotential target is selective gene silencing. This is possible
by using RNA interference (Sect.12.7). If the pinworm
(nematode) is exposed to asubstance library, it will be
possible to see achange in appearance or behavior. Is
the life expectancy lengthened or shortened? These are
indications that the compounds could interfere with the
aging process or are toxic. If there are changes in muscle
cells, perhaps it might be useful for neurodegenerative
muscle disease. Aside from macroscopic changes in the
body form, changes in the gene expression pattern can
also be analyzed (Sect.12.10). Are mutations in proteins
apparent? Certainly, the worm does not have the same
metabolic pathways as we do. Even its disease models
only partially represent the pathophysiology that is seen
in human disease. Nonetheless, direct testing of compounds on the pinworm seems to afford anew perspective for screening substance libraries. As an alternative,
the fruit y (Drosophila melanogaster) or the zebrash
(Danio rerio) are also available as test organisms. They
help to test the validity of atherapeutic approach early
in aprogram.
7.6 In Silico Screening of Virtual
Compound Libraries
As described in the previous section, experimental
high-throughput screening (HTS) has been automated
with great effort. When fed with compounds from combinatorial chemistry (Chap.11), several hundred thousand substances can be screened by using HTS. At rst,
it seemed that this would be the end of all rational structure-based techniques. In view of the enormous nancial investment and the disappointingly low hit rate, the
initial euphoria began to soberingly wane. Therefore, as
an alternative, the technique of enumerating huge databases on the computer by tting small molecules in
apredened binding pocket (docking, Sect.20.8) was
developed and referred to as virtual screening.
The unsatisfactory hit rate from HTS is attributed
to the size, structural diversity, and poorly selected composition of the substance library with respect to the actual properties of the target protein. The recognition of
false-positive and false-negative hits in biological systems
causes large problems. Disappointing hit rates have been
reported for the translation of initial hits into potential
lead structures for lead optimization. This was all the more
reason that virtual screening techniques were developed as
acomplementary and alternative method. The prerequisites for the successful use of these techniques are entirely
different from those of the technology-driven HTS: virtual
screening can only reasonably be applied if the factors that
are responsible for aputative drug to bind to its target
protein are well understood on the molecular level.
The starting point for this is the spatial structure of
the target protein, which is usually determined by NMR
spectroscopy, electron microscopy, or X-ray structure
analysis (Chap.13, . Fig.7.3). Models can be increasingly derived from structurally homologous proteins
of known geometry (Sect.20.5). It remains to be seen
whether methods based on machine learning and articial intelligence will provide sufciently accurate models
for virtual screening (Sect.20.6). To successfully bind to
aprotein, the ligand must adopt ashape that is complementary to the binding pocket. Molecules are exible and can change their shape through bond rotations
that require very little energy (Chap.16). In addition to
adopting an appropriate conformation in space to accommodate the protein binding pocket, the functional
groups of a potential ligand must nd complementary
functional groups of the protein in the binding pocket.
Hydrogen bonds must be formed between ligand and
protein, and hydrophobic molecular moieties must nd
their counterpart in the protein (Chap.4). For this, the
protein binding pocket is analyzed to highlight the regions that are essential for binding.
For aparticular atom type, for instance, ahydrogen-bond donor or acceptor, the binding pocket is
systematically scanned. By using computer graphics,
it is possible to see where functional groups attached
to a candidate ligand might be optimally placed
(Sect.17.10). The composite picture of all such placed
atom types in the binding pocket that are indicated by
this analysis reveals aspatial pattern of physicochemical properties that aligand must meet to successfully
bind to the protein (“hot spots” Sects.17.1 and17.10).
With these criteria in hand, amolecular database can
be searched that is composed of already-synthesized
compounds or compounds that have been virtually assembled on the computer. To this end, programs have
been developed that assemble huge libraries of virtual
test molecules in the computer using previously programmed synthesis rules. In case ahit from the latter
group is found, the compound can be subsequently synthesized. The search is divided into multiple ltering
steps that become increasingly stringent and sophisticated with successive reduction of the search quantity.
With the help of fast docking programs (Sect.20.8), mol-
ecules are tted into the binding pocket and abinding
geometry is generated, from which the expected binding
afnity can be estimated. This step is decisive, but unfortunately it is also the most difcult (Sect.20.9). In
Chap.21, examples that were found by avirtual screening campaign are presented.
a
f
h
. • In Silico Screening of Virtual Compound Libraries
The evaluation of the generated binding geometries
is accomplished with sufcient accuracy in about 70%
of cases nowadays. An improvement in predictive power
requires that we understand the ligand–protein recognition process better (Chap.4). The role of water in the
binding, the induced steric and dielectric adaptation,
the exible behavior and residual mobility of proteins
and bound ligands, and the dynamic changes during
complex formation are still poorly understood. The
composition of the databases themselves plays adecisive role in the search’s success. Enlarging the database
alone is not enough. The enrichment of the compounds
that could fulll the requirements is crucial. Screening is
often compared to the search for aneedle in ahaystack.
When looking for such aneedle, it is not helpful to sim-
ply double the size of the haystack! The haystack must
be spiked with more promising needles. To achieve this,
all available knowledge about the structure, function,
and dynamic behavior of the target protein must be used
to dene the database search. Comparisons between
proteins and protein binding pockets, especially among
members of the same protein family, can offer decisive
information (Sects. 20.3–20.6). In principle, all of the
data that are needed about the composition of asuitable
compound library for avirtual screening run are already
intrinsically coded in the structure and geometric interaction properties of the binding pocket. It is only aquestion of applying it correctly. Another decisive criterion
for ahit is an adequate pharmacokinetic prole so that
satisfactory bioavailability can be achieved (Chap.19).
b
c
g
. Fig. 7.3 The spatial structure of aprotein is the starting point for
virtual screening(a). The binding pocket is explored with avariety of
different probe atoms, for instance, for hydrogen-bond acceptors or
donors(b). Regions that are particularly favorable for such interacting
groups are highlighted on the computer graphics. If the “hot spots” in
these areas are summarized, aspatial pattern of properties that apotential ligand should have become apparent(c). This pattern is called
“pharmacophore” and serves as the search criterion for a database
d
e
retrieval(d). Potential ligands from alarge database are ltered and
energetically evaluated by docking(e). The discovered hits are either
commercially available or synthesized in the laboratory(f). Next, biological testing takes place(g), and if the binding is successful, the lead
structure will be crystallized with the protein. The subsequent structural determination(h) serves as a starting point for further design
cycles (for an example see Fig.02,
7 https://sn.pub/4NppHn)
Chapter • Screening Technologies for Lead Structure Discovery
7
7.7 Biophysics Supports Screening
Surface plasmon resonance (SPR) is increasingly being
used to screen for new lead structures. Atarget molecule
is anchored to the gold-coated surface of asensor chip.
Polarized light (. Fig.7.4, strictly speaking, transversely
magnetically polarized light, that is, the direction of the
magnetic eld is perpendicular to the plane spanned by
the incidence vector and the surface normal) is then irradiated from the bottom of aglass substrate. Changes in
the refractive index, which can be followed by ashift in the
angle of total internal reection, are ameasure of structural changes in the sensor surface. Changes in the masses
of bound matter also contribute signicantly to these
changes. Free electrons in the deposited gold layer interact with the incident light to form PLASMA-like electron
clouds (PLASMONS), which are nothing other than surface waves. These propagate parallel to the metal surface
and, under certain conditions (i.e., wavelength and angle of
incidence near total internal reection), can be excited to
resonance by the polarized light. This allows changes in the
refractive index in the immediate vicinity (afew 100 nm) of
the surface to be observed with very high sensitivity. For
example, if asubstance with amass greater than about 100
Da binds to the biological target molecule, the resulting
mass change can be registered on the gold surface.
There are two basic strategies for performing the measurements. In the rst, the ligand is added to the chip
with the immobilized protein in acuvette. The binding
equilibrium is established and the shifts in the reectivity
angle due to the mass effect are recorded. Adilution series is then performed. Depending on the binding afnity
of the test ligand to the immobilized target protein, its
binding behavior is evaluated from the resulting refractive index shifts. This equilibrium method has not become
widely accepted. Instead, steady-state methods are used
today. For this purpose, the test ligands are allowed to
ow over the chip carrying the protein in aconstant but,
if necessary, adjustable liquid ow. Changes in the angle
of reection are observed and converted to changes in the
refractive index. The test ligand is added to the liquid ow
in atime-controlled manner. Again, concentration series
must be performed. The latter procedure is faster to perform, but is dependent on the ow rates and, since chemical equilibrium can never be assured, assumes a“steadystate” situation. In particular, the diffusion rates must
be strictly controlled. The SPR method works fast and
atime course of the mass increase on the chip can be
observed. Thus, in addition to stoichiometry, kinetic parameters of association or dissociation become available.
Aprerequisite for using the method for screening is
stable and reproducible immobilization of the compounds
to be tested on the sensor chip. In the early days of SPR,
the sensitivity required the binding of very large masses.
Therefore, large libraries of low molecular weight test
compounds were chemically anchored to the gold chips.
The binding of amacromolecular receptor protein to the,
thus, exposed test compounds could be easily detected by
the enormous change in mass. Today, the surface plasmon
resonance method has reached such ahigh sensitivity that
even very small mass changes caused by small ligands
can be detected. For this reason, today almost exclusively
macromolecules are immobilized on the sensor chips and
the low-molecular weight test substances are allowed to
ow over these surfaces as possible binders. Since the test
substances can also be “washed off” with the liquid ow,
the “off kinetics” can be determined and the immobilized
protein can be reused several thousand times for the measurement of different test ligands.
The concept of “ligand efciency” was introduced in
Sect.7.1. To address the latter aspect, test libraries are
increasingly being supplied with compounds that have
amolecular weight of less than 250 Da. The term “chemical fragment” has become popular to refer to these search
. Fig. 7.4 Principle of surface
plasmon resonance (SPR) using
the continuous ow method. The
method registers changes in the
refractive index on the surface of
asensor chip (green). The extent
of the mass change on the gold
surface (yellow), which is caused
by binding of atest ligand ( ) to
areceptor (red ) immobilized
there, leads to ashift in the
resonance angle of the reected
light (IandII). This not only
measures the binding afnity,
it also succeeds in determining
kinetic parameters of association
(k
) and dissociation (k
on
)
off
. • Biophysics Supports Screening
. Fig. 7.5 In isothermal titration calorimetry,
asolution with aligand is added dropwise to asolution
containing aprotein. The binding to the protein leads
to an exothermic or an endothermic reaction. The heat
that evolves upon the addition of each drop is the area
under the single signal peaks. The total integral of all
signal peaks is the binding enthalpy ∆H. With increasing amount of ligand, the protein becomes saturated so
that the signal intensity of the heat signal decreases. The
binding constant (dissociation constant) can be derived
from the shape of the curve, and the free energy ∆G
can be obtained from the relationship ∆G = −RTlnKd.
The stoichiometry of the reaction is simultaneously obtained. The entropy is calculated by using the equation:
∆G = ∆H − T∆S
candidates. The term is somewhat unfortunate because
the molecules are actually “complete” small molecules
and not, as the term might suggest, simply a“fragment,”
that is an additional building block to be attached to
alead structure. In recent years, SPR has become the
standard rapid method for fragment screening.
Proteins denature when heated. A“melting temperature” is dened when an unfolding process (Sect.14.2) oc-
curs. This temperature can be measured very sensitively
with athermal sensor. The binding of aligand to aprotein changes this melting point. As described in Sect.7.3,
uorescence measurements are extremely sensitive indicators. This melting effect can be registered by allowing
the unfolded proteins to interact with auorescent dye
and detecting the change in auorescence signal. The
temperature shift caused by ligand binding can be used
as evidence of whether aligand is bound to a protein
or not. It has also been possible to construct quantitative binding assays using this effect. This highly sensitive
thermal shift method is also suitable for the detection of
weakly binding fragments.
Mass spectrometry (MS) has evolved signicantly over
the last few decades. By applying very gentle bombardment conditions, it is possible to detach single electrons
from large biomacromolecules or even to generate negatively charged species. In the best case, the protein of interest can be detected in its intact form as asingly charged
ion. The charged particles are then accelerated between
charged, parallel-oriented condensator plates. The ow
of charged particles can be bent by applying amagnetic
eld. The ight path of a given particle depends on its
mass and charge. In this way, it is possible to separate and
detect particles based on their mass-to-charge ratio. This
principle has been rened with the most sophisticated
technology and clever combination of electric and magnetic elds, so that it is now possible to detect individual
mass differences of only afew daltons among even huge
proteins. Using sophisticated experimental conditions,
agiven situation in solution, such as aprotein–ligand
complex, can be transferred to the gas phase without decomposition. There it is ionized and detected in the mass
spectrometer (Sect.21.10). With this technique, we have
an assay that can detect the binding of very small ligands
to proteins. It is even possible to tailor the decomposition
of the complexes by varying the acceleration voltage. By
registering the voltage at which decomposition occurs, the
strength of the protein–ligand complex can be assessed.
Since the decomposition occurs in the gas phase, information about the binding strength of such complexes in
awater-free environment is available.
Recently, mass spectrometry has increasingly sought
to capture spatial information about proteins in addition
to their topological composition. By inserting chemically
reactive cross-linkers between two functional groups of
a protein or protein complex, distance information in its
intact spatial structure can be captured and identied by
MS. The positions of the cross-linked amino acids, together with the length of the cross-linker, reveal distance
restraints in the 3D structure of a biomolecule. In addition, isotopic labeling by hydrogen-deuterium exchange
(HDX) can be used to determine the spatial accessibility
of main-chain amino groups. After certain time points
Chapter • Screening Technologies for Lead Structure Discovery
7
and depending on the exchange conditions applied, fast/
slow exchange in buried/accessible surface regions can
be detected by digesting and analyzing the partially labeled protein. This provides insight into which sequence
stretches have been involved in the formation of stable/
labile contact interfaces of a protein complex.
Ligands can also be “shed” with proteins. This involves exposing aprotein for which aligand is being
sought to an entire library of test compounds in aqueous solution. Those compounds from the library that
bind to the protein are captured. The protein is then
separated using amicrolter and the bound ligand is
released by chemically denaturing the protein. The solution containing the released ligands is then processed
and amicro-HPLC separation is performed. The chromatographically separated ligands are then subjected to
highly sensitive analysis to determine which members of
the original library have been shed out by the protein.
The binding of aligand to aprotein is achemical reaction. As with all chemical reactions, amore or less pro-
nounced heat of reaction can be observed. The process can
either release heat (exothermic) or absorb heat (endothermic). This heat signal can be recorded to register the binding of aligand to aprotein. Ahighly sensitive calorimeter
is required. When equipped with electronically controlled
compensation heating, these devices can achieve amazing
sensitivity. For example, such an instrument was built to
study the activity of abuttery being attracted by different pheromones. The heat generated by the wing beats was
detected as asignal by such acalorimeter.
Adissolved ligand can be titrated by dropwise injection
into the solution of atarget protein in such acalorimeter.
Each drop produces aheat signal. With increasing saturation of the protein, the heat signal decreases and acurve
can be generated from which the binding constant of the
ligand can be deduced (. Fig.7.5). From the slope of the
curve at the inection point, the dissociation constant Kd
can be determined. When all signals are integrated over
the entire titration, the total heat of reaction for the bind
ing event is determined. In this way, two different ther-
-
. Fig. 7.6 The protein to be tested is placed in acapillary with atest
ligand. Test series with several ligand concentrations are measured.
The protein has previously been covalently labeled with auorophore.
Now the infrared laser is switched on and generates alocal temperature
gradient in the region of the uorescence observation window. The
labeled molecules diffuse out of the heated region. Their migration is
tracked by the decrease in uorescence (F
migration properties depend on whether and how strongly aligand is
). The thermophoretic
norm
bound (concentration-dependent at equilibrium) to the target protein.
At the end, the laser is turned off and the system returns to the initial
state. Because the system is measured at different concentrations, the
thermophoretic effect varies in strength (i.e., from the black to the gray
and to the red decay curves) and can be converted into the shape of
abinding curve (top right). (Figure after M.Jerabek-Willemsen etal.,
J.Mol. Struct., 1007, 101–113 (2014))
. • Biophysics Supports Screening
. Fi
g. 7.7
In the switchSENSE method, ashort DNA strand is immobilized on agold surface (yellow). It carries auorophore at the end
(green ). Atarget molecule (here protein, gray) is attached to acomplementary strand that hybridizes with the rst strand. In an alternating
electric eld of varying frequency, the DNA double strand is set into
an oscillating tilting motion (purple arrow). As the strand approaches
the gold surface, the uorescence is increasingly quenched periodically.
Antibodies (schematically shown asY) are added to the test solution.
When one of these antibodies binds, the hydrodynamic frictional resistance changes. It differs whether the antibody binds to the protein ( )
or not ( ). In this way, binding can be detected and quantied. (Figure
after J.Niemax etal., Laborwelt, 11(5) 21–22 (2010))
modynamic binding characteristics are measured. The free
energy ∆G is determined from the equilibrium constant
and the enthalpy ∆H is given by the integrated heat signal
(Sect.4.3). Using Eq.4.3, the entropy of binding can be
calculated. It is important that in addition to proving the
binding of the ligand to the protein, the most relevant
thermodynamic parameters ∆G, ∆H, and ∆S can be evaluated in one experiment at one temperature.
The method also records the stoichiometry of the
binding process. Many binding processes involve changes
in the protonation state of the binding partners. This can
be recorded with the isothermal titration calorimetry
(ITC) method. To achieve this, the titration must be
performed from different buffer solutions. If the buffer
releases aproton during the binding process, its heat of
ionization must be spent for this step. Conversely, if it
captures aproton, the same energy will be released as
a heat signal. Since different buffer compounds have
different heat signatures for the protonation change, the
molar amount of protons transferred can be inferred
from this buffer contribution. In Sect.4.4 (. Fig.4.4)
such an example was presented.
Isothermal titration calorimetry is not ahigh-throughput method. Rather, it is used to analyze and characterize
the binding process. Because of its importance, especially
with respect to ligand optimization, the method will be
discussed again in Sect.8.8. Recently, the method has
been further developed by Philippe Dumas in Strasbourg, France. Kinetic data can also be derived from the
shape of the individual injection signals along the thermogram. The so-called kinITC-ETC (ETC: equilibration
time curve) is used to evaluate how quickly the thermal
signal returns to the baseline with each successive injection. This contains information about k
. For a1:1
off
binding process, the dissociation constant can be used to
infer kon, since it is the quotient of k
and kon.
off
Agroup of physicists led by Dieter Braun and Stefan
Duhr at the University of Munich in Germany has developed anew biophysical method that is now being used
worldwide for compound screening. Microscale thermo-
phoresis (MST) is apowerful technique for quantifying
biomolecular interactions. It is based on thermophoresis,
the directional movement of molecules in atemperature
gradient. This process is strongly dependent on molecular properties such as size, charge, hydration shell and
molecular conformation of the moving particles. The
technique is highly sensitive to virtually any change in
these molecular properties. However, since these properties change primarily when aligand binds to aprotein,
this approach can be used to quantify ligand binding.
This is done by measuring the system at different ligand
concentrations. Depending on the afnity and, thus, the
percentage of the bound test ligand to the target protein
in chemical equilibrium, the thermophoretic effect is different depending on the applied concentration. This can
be converted into abinding curve (. Fig.7.6).
During an MST experiment, an infrared laser is used
to abruptly create alocal temperature gradient in the test
solution (. Fig.7.6). Then, the directional movement of
molecules through the temperature gradient is recorded
and quantied. For this purpose, the protein of interest
has been covalently labeled with auorophore, or the
intrinsic uorescence of, for example, the tryptophan residues in the protein can be used. By correlating the de-
Chapter • Screening Technologies for Lead Structure Discovery
7
tected uorescence, which changes its intensity strongly
as afunction of temperature and environment, with the
variability of the thermophoretic effect, MST provides
arobust and rapid way to study molecular interactions.
The development of new uorescent dyes with improved environmental sensitivity has made the MST
method even more sensitive. In particular, the temperature-dependent intensity change in the induced temperature gradient is exploited. As aresult, it is now possible to
perform the method in 384 microtiter plates. This opens
the door to HTS; aplate can be screened in about 30 min.
Another relatively new method is the switchSENSE
technology developed by Dynamic Biosensors, led by Ulrich Rant of Munich, Germany. In this technology, ashort
strand of DNA is anchored to agold surface (. Fig.7.7).
On the opposite end, this strand carries a uorescent
probe. It can be hybridized to asecond strand of complementary sequence. This second strand carries achemical
functionality on its side facing away from the gold surface
so that atarget molecule can be attached to it. Because
the strands are made of DNA, they carry many negative charges. When an alternating electric eld is applied
across the gold surface, the DNA strands can be stimulated to oscillate towards and away from the gold surface.
This oscillation can be tracked in real time because the
uorescence of the attached uorophore is quenched as
it approaches the metal layer, which has an increasing and
decreasing positive charge in the alternating eld. Thus,
the position of the DNA strands in their switching motion
can be observed by the intensity of the uorescent light.
The dynamics of the switching motion pull the molecule
of interest through the solvent. Hydrodynamic friction
and the charges on the target molecule inuence the tilting
motion. If aligand is bound to the target molecule, or if
aconformational change is induced for some reason, the
switching motion can be slowed down. This is used as
the measurement signal of the assay. This process can be
analyzed very precisely by tuning the alternating eld in
afrequency-dependent manner. Thus, in addition to the
binding afnity, binding kinetic parameters such as kon
and k
become available. . Fig.7.7 shows an example
off
of the binding of an antibody to aprotein. The method
can be extended in many ways to molecular systems,
provided they can be chemically immobilized on ashort
DNA strand. For an assay developer, there are almost
no limits to the measurement setups that can be created.
7.8 Screening by Using Nuclear Magnetic
Resonance
The method of NMR spectroscopy is described in more
detail in Sect.13.7. Sufce it to note here that it is concerned with the orientation of the magnetic moments of
the nuclei in asample in amagnetic eld. By applying
acarefully chosen, spatially and temporally resolved sequence of electromagnetic elds, it is possible to specifically activate nuclei that are oriented within these magnetic elds. This can be done, for example, for one type
of nucleus in aprotein. If asolution of test ligands or
awhole mixture of ligands is added to such asolution,
protein binding can occur, provided that the ligands are
suitable. Depending on their binding strength, they will
remain on the magnetically saturated protein for acertain
. Fig. 7.8 To determine the saturation transfer difference (STD)
with NMR spectroscopy, alibrary of test ligands ( , ) is added to
atarget protein (ellipse). Potential binders (here ) reside for anite
time span bound to the protein. If the nuclear spin of one type of
nucleus in the protein is selectively saturated (red) by using asuitable
resonance frequency (RF), the protein magnetization will be transferred (nuclear Overhauser effect, see Sect. 13.7) to the ligand that
was bound in the meantime ( ). These ligands become apparent in
that their spectrum is altered even though they are already dissociated
from the protein. If the difference between the spectra in presence of
the saturated and unsaturated protein is displayed, it will be possible
to determine which ligands were bound immediately to the protein.
Many variations and sophisticated experimental protocols have been
developed for the principle of magnetization transfer