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. • What Is the Contribution of aHydrogen Bond to the Strength of Protein–Ligand Interactions?
. Fig. 4.11 Fidarestat4.6(a) forms ahydrogen bond with its carbox-
amide group to the NH group of Leu 300. By exchanging leucine for
proline, the H-bond can no longer be formed. This leads to a∆∆G
loss of 7.8 kJ/mol, which is essentially being paid for by an enthal-
Pro
pic price (∆∆H
to aldose reductase via three H-bonds with its carboxylate group. If
Tyr48 is exchanged for Phe48, a charge-assisted contact is lost at
this site. The loss in ∆∆G
ample. However, it partitions into a huge enthalpic loss and smaller
entropic gain.(c) In this example, the ligand4.8 with a phenyl ring
was modied into the benzamidine analogue4.9. Asalt bridge to Asp
189 in thrombin is additionally formed, resulting in astrong gain in
: 6.9 kJ/mol). The inhibitor IDD5944.7(b) binds
Leu⇨Pro
is slightly larger than in the rst ex-
Tyr⇨Phe
in the inventory. The proline mutant cannot establish
awater-mediated contact with sorbinil due to the lack
of an NH function. Therefore, the enthalpic gain due to
the H-bond is missing. However, there is also no entropic
∆∆G
In sorbinil 4.10(d), the carboxamide group is lacking compared to
darestat4.6. Considering again the exchange Leu⇨ Pro in aldose
Leu⇨-
reductase, this leaves the free binding energy ∆∆G
changed. However, sorbinil binds to the wildtype enzyme (leucine)
enthalpically more favorably and entropically less favorably than to
the proline variant. An interstitial water molecule mediates an H-bond
between sorbinil and Leu 300, yielding an enthalpy advantage of
−5.1 kJ/mol here. Simultaneously, however, the entrapment of awater
molecule is entropically unfavorable (–T∆∆S
virtually compensates the entire enthalpic advantage
. This is governed by an enthalpic gain; entropy is opposing.
4.8⇨4.9
Leu⇨Pro
: 5.9 kJ/mol) and
Leu⇨Pro
this charged amino acid in asalt bridge, 4.11 was terminally carboxylated to 4.12. Although the crystal structure
shows that the proposed salt bridge is indeed formed, its
contribution to the afnity increase of 4.12 over 4.11 is
loss because no water molecule is entrapped.
The three-dimensional structure has been determined
for alarge number of protein–ligand complexes. Many of
these complexes form hydrogen bonds between protein
and ligand. The whole issue concerning the contribution
of ahydrogen bond to binding afnity becomes evident in
. Fig.4.12. Here, for arandom selection of 80protein–
ligand complexes, the experimentally determined binding
constants (logarithmic scale) are plotted against the number of hydrogen bonds. For agiven number of hydrogen
bonds, the measured binding constants cover aconsiderable range. Thus, the contribution of an H-bond is by no
means constant, but varies considerably. Due to unfavorable desolvation effects, the contribution of an H-bond
can even decrease the binding afnity.
Afurther important contribution to acharge-assisted
hydrogen bond is where it is formed in the protein complex. In . Fig.4.11c, asalt bridge makes alarge contribution to the transition from4.8 to4.9. Deep in the
S1 pocket of thrombin, the benzamidine group of4.9
forms asalt bridge with the carboxylate group of Asp
189 (. Fig.4.13, left). In the enzyme tRNA-guanine
transglycosylase (Sect.21.9), the binding mode of inhib-
itor 4.11 was characterized (. Fig.4.13, right). It carries
aterminal phenyl ring, but is spatially close to the amino
acid Arg 286 as seen in the crystal structure. To involve
. Fig. 4.12 A logarithmic plot of the binding constant of 80 pro-
tein–ligand complexes studied crystallographically against the number of hydrogen bonds formed between protein and ligand shows that
there is no simple and direct correlation between the two properties
virtually un-
4
Chapter • Protein–Ligand Interactions as the Basis for Drug Action
. Fig. 4.13 For the contribution of a salt bridge to binding afni-
ty, the environment in which this salt bridge is formed is crucial. In
thrombin (left), the introduction of a benzamidine group 4.8⇨4.9
deeply buried in the S1 pocket leads to the formation of asalt bridge
with Asp 189. The afnity gain is very large (see . Fig.4.11c). In the
tRNA-guanine transglycosylase (right), the addition of acarboxylate
group to the terminal phenyl ring 4.11⇨4.12 does not enhance the afnity of the nanomolar binding inhibitor 4.11. The crystal structure
proves that the salt bridge to Arg 286 is formed geometrically. Howev-
very small. The introduced salt bridge remains largely
exposed to the surrounding solvent water, which reduces
its impact on the afnity of the interaction.
An impressive example of the importance of hydrogen bonds is provided by the inhibitors 4.13 of the
metalloprotease thermolysin, synthesized in the research
group of Paul Bartlett. Aphosphonamide –PO2HN– was
replaced by aphosphinate –PO2CH2– or aphosphonate
–PO2O–. The results of these exchanges are summarized
in . Table4.3. Although the X-ray structure shows that
the NH group forms an H-bond to the carbonyl oxygen
of Ala 113, it can be replaced by aCH2 group without
loss of binding afnity. This result may be surprising
at rst glance, but it can be understood by comparing
the number of hydrogen bonds before and after ligand
binding for the phosphonamide and phosphinate, analogous to . Fig.4.6. The ligand forms an H-bond to
water molecules via its NH function before binding to
the protein in aqueous solution. The ligand has to aban-
er, since it remains largely exposed to the surrounding solvent, its contribution is only about one tenth of the buried salt bridge in thrombin.
(7 https://sn.pub/BJz11h)
don this H-bond when it enters the binding pocket, but
there it forms acomparably strong interaction with Ala
133(C=O). Thus, the ligand loses one hydrogen bond but
gains another; hence, the inventory is balanced. In the
phosphinate, the –CH2– group cannot form an H-bond
in water prior to protein binding. No H-bond is formed
in the binding pocket either. Again, the inventory is balanced. In both cases, the number of H-bonds remains
the same. If the NH group is replaced by an oxygen
atom, the binding afnity decreases by afactor of 1000.
In water, the oxygen atom that replaces the NH group
can form ahydrogen bond with the bulk water. In the
protein–ligand complex of the phosphonate –PO2O–,
the electronegative oxygen atom is exactly opposite the
oxygen of the carbonyl group of Ala 113. Two acceptor groups face each other. Ahydrogen bond cannot be
formed here. The hydrogen bond inventory remains un-
balanced. In addition, the two groups repel each other,
resulting in weaker binding.
. • The Strength of Hydrophobic Protein–Ligand Interactions
. Table 4.3 Binding constants Ki for the thermolysin inhibi-
tors 4.13, which contain either aphosphonamide (X = –NH–),
aphosphonate (X = –O–), or aphosphinate (X = –CH2–)
group. The phosphonamide group –PO2NH– complexes the
zinc ion and simultaneously forms an H-bond with Ala113
R Binding constant Ki in μM
X = –NH– –O– –CH
OH 0.76 660 1.4
Gly–OH 0.27 230 0.3
Phe–OH 0.08 53 0.07
Ala–OH 0.02 13 0.02
Leu–OH 0.01 9 0.01
–
2
Asimilar case is illustrated in . Table4.4. Here the
binding afnity of three thrombin inhibitors 4.14 that
were synthesized at Eli Lilly are compared with each
other. The amine (X = –NH–) can form an H-bond
with Gly 219 and binds the most strongly. The ether
(X = –O–) binds 5000-times weaker because of an electrostatic repulsion between the ether oxygen atom and
the carbonyl group of the protein. The aliphatic compound (X = –CH2–) shows remarkable binding compared to X = –NH– that is merely reduced by afactor
of eight (thrombin) and two (trypsin).
. Table 4.4 Binding of 4.14 to the serine proteases thrombin
and trypsin
Enzyme IC
Thrombin 0.009 52 0.07
Trypsin 0.009 43 0.018
values in mg/mL
50
X = –NH– –O– –CH
–
2
4.9 The Strength of Hydrophobic
Protein–Ligand Interactions
We have seen that the direct attractive forces between lipophilic groups are much weaker than those between polar groups. Hydrophobic interactions are based on weak
attractive forces in the immediate molecular environment
(Sect.4.4). They are formed in the protein environment
as well as in the aqueous milieu. Therefore, they contribute little to the inventory. For hydrophobic interactions,
however, it is mainly the displacement and rearrangement
of the neighboring water molecules that counts. It has
been shown in many experiments that their contribution
to the binding afnity is, to arst approximation, proportional to the size of the lipophilic surface that is buried
upon ligand binding and therefore no longer accessible
to water. Typically, the contribution is found to be in the
range of −50 to −200 J/mol per Å2 of lipophilic contact
. Fig. 4.14 The scaffold of the HIV protease inhibitor 4.15 was en-
larged during the course of a lead structure optimization by adding
hydrophobic groups to the aromatic N-benzyl group. An unchanged
binding mode was evidenced crystallographically. The additional molecular volume improved the binding afnity in alinear manner by
about −65 J/molÅ
2
4
Chapter • Protein–Ligand Interactions as the Basis for Drug Action
. Fig. 4.15 In analogy to . Fig.4.12, alogarithmic plot of the bind-
ing constants Ki of the 80crystallographically investigated protein–ligand complexes against the buried hydrophobic surface area shows
that there is no simple function for this measure either
area. An example of this is retinol. It binds to the retinol binding protein (. Fig.4.1) with abinding constant
of 190 nM exclusively through lipophilic contacts. This
corresponds to afree energy of −39.8 kJ/mol. As aresult
of the binding, alipophilic area of 250 Å2 is buried. The
contribution per Å2 is −39,800/250 = −159 J/molÅ2.
Six HIV protease inhibitors (Sect.24.6) are listed in
. Fig.4.14. During the course of alead structure optimi-
zation, the hydrophobic surface of 4.15 was enlarged by
adding hydrophobic groups. It could be conrmed crystallographically that the binding mode did not change.
If the variations in the molecular volume in this series
are plotted against the afnity, alinear relationship is
obtained. The binding afnity increases by −65 J/molÅ2.
In many cases, the hydrophobic interactions are
adominant contribution to the free energy of binding.
In . Fig.4.15, the lipophilic surface area that is buried
upon complex formation of the same 80protein–ligand
complexes as in . Fig.4.12 are shown together with their
experimentally determined binding constants. Here too,
the values are scattered over abroad range.
4.10 Binding and Mobility: Compensation
of Enthalpy and Entropy
According to Eq.4.4, enthalpy and entropy have aclose
physical relationship and their combined contributions
give the Gibbs free energy of binding. Considering
the formation of protein–ligand complexes, the ∆G of
weakly binding millimolar complexes and strongly bind-
ing nanomolar complexes are in the range of about 35–
55 kJ/mol. Lead optimization (Chap.8) usually covers
an even smaller range. Typically, the binding constants
are improved by 5–6 orders of magnitude, corresponding
to 25–30 kJ/mol. When functional groups are exchanged
in alead structure, the change in enthalpy ∆H usually
varies over amuch larger range. If the variation of ∆G
is much smaller for this transformation, the change in
enthalpy ∆H has to be compensated by achange in entropy −T∆S in opposite direction, simply because of numerical reasons. Only in this way can the large variations
in the two properties lead to the result that ∆G remains
in asmall window. This leads to an important question:
Is there aconnection that causes the opposing enthalpy
and entropy to partially compensate during optimization?
If there is compensation, how can both quantities be
optimized without canceling each other out, so that ∆G
remains unchanged?
Entropic optimization is aimed at increasing the hydrophobic surface area buried upon binding. This very
descriptive quantity expresses that enlarged ligands displace an increasing number of water molecules upon
binding. In particular, if these displaced water molecules
were previously well xed in the binding pocket, an entropically favorable signal will result.
The entropic contribution to binding of aligand can
be enhanced by synthesizing compounds that have reduced degrees of freedom around bonds, while maintaining ageometry that ts into the protein binding pocket.
For example, in the peptide-like thrombin inhibitor 4.16,
asubstituted glycine residue in the center was exchanged
for the more rigid proline to form 4.17. Crystallographically, it was found that the binding mode of both ligands
remained unchanged. The rigidized ligand4.17 is more
potent by −10.8 kJ/mol (about two orders of magnitude
in binding constant!), which can be attributed to afavorable entropic binding contribution. Molecular dynamics
simulations of both ligands in aqueous medium prior to
protein binding show that the accessible conformational
space of the proline derivative 4.17 is signicantly restricted compared to the open-chain glycine derivative
4.16. This has astrong impact on the number of degrees
of freedom that are sacriced during binding.
The two thrombin inhibitors 4.18 and 4.19 were synthesized with the same goal. In their case, the rigidication should be realized through an intramolecular hydrogen bond to the hydroxymethylene and aminomethylene
anchors on the phenyl ring, respectively. First, the two
compounds were shown to adopt an identical binding
mode in the thrombin binding pocket. The observed geometry also corresponds to the binding mode adopted
by inhibitors that completely lack the anchor. But to
what can the increase in afnity from 4.18 to 4.19 then
be attributed, since both compounds form the desired
stabilizing H-bond in the binding pocket? The difference
is to be found in the rigidication of the ligands in aque-
. • Binding and Mobility: Compensation of Enthalpy and Entropy
. Fig. 4.16 Effect of rigidication or “pre-organization” of inhib-
itors on protein binding. Left The peptide-like thrombin inhibitors
4.16 and 4.17 differ only in the exchange of acentral substituted glycine residue for proline. This conformationally restricts the molecular scaffold of 4.17 to afew conformers that are also adopted at the
binding site. 4.17 therefore sacrices fewer degrees of freedom upon
binding than 4.16 and binds with adistinct entropic advantage. Right
In thrombin inhibitors 4.18 and 4.19, an anchor was introduced to
stabilize the ligands in the bound state by an intramolecular H-bond.
ous solution before binding to the protein. Whereas the
aminomethylene derivative 4.19 is stable with an intramolecular hydrogen bond even before protein binding,
the H-bond in the hydroxymethylene analogue 4.18 is
unstable. In the unbound state, there is hardly any formation of the conformation of this ligand taken up in
the protein. Only if this is achieved does rigidication
of aligand lead to the expected entropic gain in afnity
(see . Fig.4.16).
In order to enthalpically increase the binding afnity
of aligand to aprotein, it is necessary to introduce additional polar interactions. However, this usually comes
at the cost of the additional polar groups having to shed
their water shell. This contribution to ligand desolva-
tion must be provided. . Fig.4.11c compares thrombin
inhibitors4.8 and4.9. Here, the added amidino group
in4.9 allows asignicant increase in afnity with alarge
increase in enthalpy. However, the desolvation of the
charged inhibitor must be overcompensated. The cost
of desolvation can be very high, as shown by the comparison of thrombin inhibitors 4.20 and 4.21 (. Fig.4.17).
Crystallographically, the same binding mode is found
for both. However, the afnity of 4.21 with the charged
pyridinium group is 11 kJ/mol lower than that of the uncharged isostere 4.20. This is due to the high enthalpic
cost of shedding the solvate shell around the charged
pyridinium derivative.
The binding mode adopted is similar to that present even in inhibitors
that lack this anchor. However, in solution prior to binding, the intramolecular H-bond to the aminomethylene group is stable, whereas it is
not in the case with the hydroxymethylene group. Therefore, only 4.19
is correctly “pre-organized” for protein binding. Here, rigidication
results in the desired entropic gain. For 4.18, this advantage is lacking
as the compound adopts the required conformation only after accommodation in the protein binding pocket
Afurther example of the importance of the contribution of the ligand dynamics of an interaction is
demonstrated with the thrombin inhibitors 4.22 and
4.23 (. Fig.4.17). They differ only in the size of their
terminal cycloalkyl residue, which was added to the parent scaffold to ll the hydrophobic S3/S4 pocket of the
protein. Both inhibitors have virtually the same binding
afnity for thrombin. However, their free binding energy
decomposes very differently into contributions from enthalpy and entropy. The compound with the cyclopentyl
substituent has an enthalpic advantage and an entropic
disadvantage compared to the six-membered ring derivative.
What is the reason for this surprising effect of the shift
in the enthalpy/entropy partitioning? The crystal structures
of both derivatives with thrombin show an important difference with respect to the terminal cycloalkyl substituent. While the ve-membered ring is well observed in
the electron density (Sect.13.5), virtually no density can
be detected in the region where the six-membered ring
should be found. Such an observation in acrystal structure indicates increased disorder of aparticular structural
building block in aprotein–ligand complex. This disorder may be purely static, in which case the six-membered
ring is scattered over many arrangements. Alternatively,
for dynamic reasons, it may have amuch higher residual
mobility in the protein-bound state than the ve-mem-
4
Chapter • Protein–Ligand Interactions as the Basis for Drug Action
. Fig. 4.17 Left Replacement of the meta-tolyl substituent in the
thrombin inhibitor 4.20 with an isosteric methylpyridium group in
4.21 results in asignicant loss of afnity, essentially at the price of
enthalpy. The charged head group for the S1 pocket of thrombin requires avery high price of desolvation but does not allow additional
favorable interactions in the enzyme. Right The homologous ligands
bered ring derivative. Molecular dynamics simulations
(Sect.15.7) conrm this latter difference due to different
residual mobility. In the case of the ve-membered ring
compound, the cyclopentyl moiety remains in ahydrophobic pocket and performs aso-called jump rotation
from time to time. In this process, the planar ring swaps
between two congurations, exchanging its top and bottom faces. But the overall position and occupancy of
the ring in the pocket remains virtually unchanged. Ligand4.22 does not form ahydrogen bond (Sect.23.4) to
the carbonyl group of Gly 216. The six-membered ring
derivative 4.23 behaves quite differently. Here, the cyclohexyl substituent moves out of the binding pocket during
the course of the simulation and returns back to it after
some time. At the same time, 4.23 forms an intermediate
hydrogen bond to Gly 216. Thus, 4.23 has ahigh residual
mobility in the bound state and scatters over several spatially distinct arrangements.
This difference in the dynamic behavior of 4.22 and
4.23 explains their deviating thermodynamic proles.
The cyclopentyl derivative has an entropic disadvan
tage because it binds more tightly in the binding pocket.
However, the uniform orientation gives this ligand an advantage in forming stable enthalpically favorable interactions with the protein. The situation is different for the
six-membered ring derivative. Its reduced spatial xation
in the binding pocket is accompanied by asmaller loss of
degrees of freedom in complex formation. This implies an
entropic advantage. Enthalpically, however, this behavior
is disadvantageous. Due to the intermediate leaving of
the ligand from the binding pocket, interactions with the
protein can only be formed with reduced strength.
What can we learn from this example? Even if ligands
have avery similar chemical structure, their binding be-
4.22 and 4.23 bind equally strongly to thrombin, but the binding afnities partition quite differently into enthalpic and entropic contributions. 4.23 has much higher residual mobility in the binding pocket
than 4.22, giving this derivative an entropic advantage. But because of
the poorer contacts to the protein on average, an enthalpic disadvantage results
havior can differ signicantly. Their residual mobility in
the binding pocket can be decisive for the thermodynamic
binding contributions. Obviously, amutual compensation
of enthalpy and entropy leads to an almost unchanged
free energy ∆G. This interplay between residual mobility
in the binding pocket and the quality of the interactions
formed clearly has consequences for the optimization
process and is one explanation why enthalpy and entropy
often compensate in optimization.
Medicinal chemists like to think in terms of standardized group contributions that the exchange of cer-
tain moieties and functional groups at agiven scaffold
might provide to the binding afnity. Statistical analyses
of such group contributions have been performed and
can be used as aset of rules for optimization strategies.
Mostly, these rules are considered as additive. How much
is gained by combining aparticular group with another
on amolecular scaffold to be optimized? However, care
must be taken when applying such considerations. Small
differences in binding behavior often cause these simple
rules to break down.
As an example, consider the optimization of thrombin
inhibitor 4.24 to 4.25 (. Fig.4.18). Two chemical modications will be performed. In the rst step, the terminal
hydrophobic substituent at one end of the parent scaffold
is increased from an n-propyl to aphenylethyl substituent.
This results in asignicant increase in the hydrophobic
surface area of the molecule, but the binding afnity is
only slightly improved by ∆∆G = −3.1 kJ/mol. As asecond
subsequent optimization step, an amino group is introduced adjacent to the hydrophobic group to form ahydrogen bond to Gly 216. This adds another −15.5 kJ/mol,
so the two modications from 4.24 to 4.25 result in an
afnity increase of ∆∆G = −18.6 kJ/mol. Does the addi-
. • Binding and Mobility: Compensation of Enthalpy and Entropy
. Fig. 4.18 Optimization of thrombin inhibitor 4.24 to 4.25 yields
an afnity increase of ∆∆G = −18.6 kJ/mol (diagonal arrow). This is
achieved by increasing the hydrophobic side chain (red) from n-pro-
pyl to aphenylethyl residue and adding an amino group (blue). The
changes can also be performed stepwise. Increasing the hydrophobic
surface area to 4.26 improves the afnity by only −3.1 kJ/mol (top ar-
row). Amajor contribution of −15.5 kJ/mol is provided by the subsequently introduced amino group (right arrow downwards). The ligand
tional amino group add that much to the afnity, and can
we include this value in our list of rules for standardized
group contributions? Of course, as avalidation, the two
modications can be introduced in reverse order via intermediates 4.26 and 4.27, respectively. If the reverse route is
followed and the amino group is introduced rst at 4.24 to
4.27, the gain in ∆∆G = −9.6 kJ/mol, asignicantly smaller
contribution by this group than along the rst route. The
subsequent increase of the hydrophobic surface from 4.27
to 4.25 now achieves an additional afnity gain of −9.0 kJ/
mol. This now asignicantly larger contribution for the
hydrophobic group than along the rst synthesis path.
This example shows that simple additivity rules of
standardized functional group contributions fail. Instead
cooperativity matters. As in the example with the veand six-membered ring derivatives 4.22 and 4.23, the
balance between residual mobility, partial solvation of
the binding pocket and the quality of the interactions
formed has adecisive inuence on the afnity increase.
The interplay of partially compensating enthalpic and en-
tropic binding contributions is responsible for this complex picture. The bulky phenylethyl substituent xes 4.26
forms acharge-assisted H-bond to the protein via this group. Reversing of the synthesis steps by adding the amino group rst to 4.27 yields
−9.6 kJ/mol (left arrow downwards), and the subsequent substitution
of the hydrophobic portion enhances the afnity by afurther −9 kJ/
mol (bottom arrow). The reason why standardized group contributions
cannot simply be added up here lies in the complex interplay of residual mobility, desolvation, and strength of the enthalpic interactions
that are formed
quite well in the binding pocket, whereby the ligand gains
hydrophobic interactions and displaces water molecules
from the protein pocket. However, it pays entropically
for the loss of residual mobility compared to 4.24, so the
gain in afnity due to the increased size of the hydrophobic substituent is only moderate. However, once this price
is paid, the additional charge-assisted H-bond of 4.26 ⇨
4.25 provides alarge amount of afnity gain. Following
the reverse optimization strategy, the H-bond introduced
by 4.24 ⇨ 4.27 now has to pay part of the price for the
reduced residual mobility. Therefore, the contribution of
the H-bond is now much smaller. Once the ligand4.27 is
spatially xed in the pocket, much more afnity can be
gained by attaching the larger hydrophobic substituent to
4.25. The price of the entropic loss of degrees of freedom
has already been paid.
This example illustrates the dilemma that medicinal
chemists often face when optimizing lead structures, as
expectations of success on the way to the planned end
product, which are usually based on estimates from standardized group contributions, can be disappointed already after the rst synthesis steps. If we were to take the
Chapter • Protein–Ligand Interactions as the Basis for Drug Action
4
path from 4.24 to 4.27, we would likely be much more optimistic about achieving the optimization goal than if we
were to go from 4.24 to 4.26, because the rst step in the
latter synthesis route yields amuch smaller afnity gain.
At rst glance, enthalpy/entropy compensation appears
to be an unavoidable curse that can easily thwart amedicinal chemist’s optimization efforts. However, decomposing afnity as afree energy quantity into entropy and
enthalpy provides deeper insight into the mechanisms that
ultimately enable potency enhancement. However, it must
never be forgotten that the entire process must be considered! Everything has to be taken into account, from the
ligand and protein in the aqueous solution before binding,
including all the water molecules involved, to the formed
complex with its newly created solvation shell. Elaborate
microcalorimetric analyses condense this complex process from amultitude of steps to three numerical values.
In the end, they represent the thermodynamic prole of
the entire binding event. Whether the result is amore
enthalpy- or entropy-driven binding is determined by the
overall balance. Michael Gilson and his group coined the
term enthalpy–entropy transduction to describe the conse-
quences of local perturbations in protein–ligand binding
that, for example, mask the enthalpic binding of aligand
to its protein. The enthalpic signature of binding can be
obscured by the thermodynamics of aglobal conformational change during complex formation. If it is induced
as aconsequence of the binding of the ligand, it may be
accompanied by achange of the system under consideration to aconformational state of higher entropy. It is
possible that such considerations help to explain why proles with pronounced enthalpy–entropy compensation are
observed experimentally in so many binding events. This
masking could produce amisleading picture of the actual
driving forces of binding. This makes it all the more important to always compare systems relative to each other
by varying only one or afew parameters or properties.
4.11 Lessons for Drug Design
This chapter is not intended to leave the impression
that protein–ligand interactions are too complicated for
quantitative predictions of the strength of protein–ligand
interactions. Rather, quantitative correlations can be established. All the more, some general rules or guidelines
should be followed when optimizing alead structure:
Many strong protein–ligand interactions are char-
-
acterized by extensive lipophilic contacts. Increasing
the lipophilic contact area between the protein and
the ligand often leads to an improvement in binding
afnity largely as the result of entropic gains from the
release of ordered water molecules from the surfaces.
This means that the search for unoccupied lipophilic
pockets in the protein should be the rst step in the
design and optimization of new ligands. However,
this approach should not be taken too far, as alarge
increase in the overall lipophilicity of amolecule will
increasingly reduce its water solubility.
The binding prole for the release of a water molecule
-
can range from dominantly entropic to dominantly
enthalpic. The release of xed water molecules usually
correlates with an entropic signature, while the release
of highly dynamic water molecules correlates with an
enthalpic signature. Most likely, the whole range from
more entropic to more enthalpic dominated signature
for ∆G can be found, but with the drawback that the
contribution of water displacement to the binding free
energy of ligand binding is increasingly offset by mutually compensating effects. This is particularly the
case when the water displacement prole is characterized by a balanced enthalpy/entropy signature. Neutron diffraction has shown that the residual mobility
of water molecules involved in ligand binding may
differ from ligand to ligand, making the estimation
of the water signature even more complex.
An increase in binding afnity due to additional
-
H-bonds is not guaranteed. For the overall afnity contribution, an H-bond will only have asignicant effect
if the interactions of the H-bonded groups in the protein–ligand complex will be stronger than in the aqueous
environment with the surrounding water molecules. In
general, such an H-bond provides amuch stronger afnity contribution when it is shielded from solvation
and formed in adeep binding pocket. This is especially
true when the binding partners carry aformal charge
and charge-assisted H-bonds are formed. An optimal
situation can be expected when the involved functional
groups undergo apKa shift due to mutual polarization,
transforming groups that were uncharged in aqueous
solution prior to binding into charged groups in the
protein binding pocket. This requires asophisticated
adjustment of pKa values in the designed molecules.
On the other hand, burial of polar atoms without sat-
-
urating them with an H-bond almost always results in
aloss of binding afnity. Ligand design must ensure
that polar ligand atoms nd binding partners in the
protein when they are no longer accessible to water
in the formed protein–ligand complex.
Aligand almost always displaces water molecules during
-
protein binding. There are protein binding pockets that
are designed in such away that they cannot be optimally solvated by water for geometric reasons. In these
cases, aligand may be able to form more H-bonds
to the protein than water molecules can. The binding
afnity of such ligands can be very high.
However, hydrophobic binding pockets can also be
-
lled with very few or even no water molecules in
the uncomplexed state. In such pockets, hydrophobic
groups achieve very strong enthalpic binding.
Rigid, properly “pre-organized” ligands can bind more
-
tightly than exible ligands because the loss of inter-
. • Synopsis
nal degrees of freedom is less for rigid ligands. It is
important to note that the “pre-organized” geometry
must already be conformationally populated in aque-
ous solution prior to binding.
When aligand occupies binding sites that are open
-
to the solvent, its substituents can contribute to the
new surface of the formed protein–ligand complex.
If they allow the formation of an optimally structured
solvent shell with fused H-bonded rings of water mol-
ecules covering the ligand portions oriented towards
the outside of the protein pocket, the ligand will gain
binding afnity. In contrast, polar groups in this in-
terface region can disrupt the solvation pattern and
remain insufciently solvated themselves. This will
result in aloss of afnity.
Placement of atitratable functional group of aligand
-
in aprotein binding pocket can result in local pKa
shifts of several log units. Ideally, this leads to the for-
mation of a charge-assisted H-bond to the protein,
whereas this group is previously uncharged in the
aqueous medium or during membrane passage. Its
desolvation is more favorable, and the charge-assisted
H-bond formed improves afnity. Charge-assisted
H-bonds make agreater contribution to afnity when
formed in deep pockets shielded from solvent access.
By clever design, ligands in the complex can adopt
-
abinding geometry in which they shield their charge-as-
sisted H-bonds to the protein themselves from the sur-
rounding solvent. This can greatly increase afnity.
If two ligands that bind independently to the protein
-
can be successfully merged or fused into one molecule
without much perturbation of their binding modes,
astrong gain in afnity can be expected. Such con-
cepts can be applied especially in the early stages of
fragment-based lead discovery (Sect.7.9).
The relative contributions of changes in enthalpy and
entropy to the binding afnity are of great importance.
The combination of these terms results in ∆G which is
the overall quantity that is optimized in adrug design
project. This goal can be achieved by improving either
the enthalpic or the entropic contribution. Ideally, both
quantities are optimized simultaneously. However, this
requires focusing on different parameters of the protein–ligand interactions (Sect.8.8). An open question
is whether a more enthalpically or entropically driven
binding will provide advantages for the optimization of
aparticular drug. The strategy to be pursued will depend
on whether tolerance to rapidly emerging resistance mutations (Sects.24.5, 31.5, 32.5) is desired for the binding
of the drug to be developed. In other cases, high target
selectivity or broad binding promiscuity within aprotein
family may be desired for an optimal therapeutic effect
(Sects.25.6, 26.4, 27.5). Ultimately, these criteria determine whether amore enthalpically or entropically driven
binding is the better choice in agiven drug design project.
4.12 Synopsis
Emil Fisher introduced the “lock-and-key” principle
-
to describe the interaction of asmall-molecule substrate and amacromolecular receptor. More than
50years later, Koshland extended this picture by induced-t considerations that allow both binding partners to change conformations and mutually adapt to
each other to optimally interact.
The cells are surrounded by alipid double-layer mem-
-
brane with polar head groups on the exterior and
hydrophobic alkyl chains in the interior. This membrane is abarrier for polar substances, but sufciently
lipophilic compounds can penetrate and even pass
through the membrane.
The strength of protein–ligand interactions is mea-
-
sured by the binding constant, which quanties the
stability of aprotein–ligand complex as adissociation
constant according to the law of mass action for complex formation.
The binding constant is logarithmically related to the
-
change in Gibbs free energy of binding. The change
in free energy is composed of an enthalpic and entropic contribution. The enthalpic part summarizes
all terms that relate to the interaction energy of the
binding partners. The entropic part considers the ordering of the system and how its energy content is
distributed over the degrees of freedom of the system.
Protein–ligand complexes usually form through non-
-
covalent interactions, predominantly through hydrogen bonds. The strength of hydrogen bonds strongly
depends on the distributions of charges among the
interacting functional groups. Whether a group is
charged or not depends on its protonation state,
which is dened by the pKa value of the titratable
groups involved in the protein–ligand interactions.
Depending on the local environment in a bind-
-
ing pocket, the pKa values of titratable groups can
vary signicantly and can, by this, transform anormal H-bond into amuch stronger charge-assisted
H-bond.
Hydrophobic interactions form through the close
-
proximity of nonpolar functional groups of the binding partners. As direct interactions, they are rather
weak. Nevertheless, they can afford asignicant contribution to binding afnity through the release of
water molecules from either the lipophilic environment of the binding pocket or from the ligand surface
next to alipophilic surface patch.
The strength of protein–ligand interactions is
-
strongly inuenced by the water environment. Both
the protein binding pocket and the ligand are solvated
before complex formation and functional groups of
the protein and ligand will form H-bonds to water
molecules. The total balance of the hydrogen-bond
Chapter • Protein–Ligand Interactions as the Basis for Drug Action
4
inventory before and after complex formation matters
for binding afnity considerations. Only if the num-
ber of newly formed hydrogen bonds in the complex
is increased in number and/or strength compared to
those previously formed in the bulk water phase will
anet afnity increase result.
The release of water molecules from hydrophobic sur-
-
faces can increase afnity by enthalpy and entropy.
Release of xed water molecules increases the de-
grees of freedom and boosts entropy. Replacement of
highly disordered water molecules into the bulk water
environment can contribute to an enthalpic gain.
Entropic contributions to binding arise from an in-
-
crease of the degrees of freedom of the protein–li-
gand–water system and, as arst approximation, cor-
relate with the size of the hydrophobic surface buried
in the formed complex.
At the end of complex formation, the ligand forms
-
anew surface with the protein which is solvated. If
optimal water networks can form around the exposed
parts of the ligand that participate in the shared sur-
face, the bound ligand gains additional afnity.
Correct rigidication (“pre-organization”) of aligand
-
into the conformation required at the binding site can
lead to asignicant increase in afnity for entropic
reasons. Importantly, however, the pre-organized
conformation must be populated in aqueous solution
prior to protein binding.
Change in free energy variations are observed over
-
a range of about 15–60 kJ/mol in protein–ligand
complexes. Variations in enthalpy (∆H) and entropy
(−T∆S) can be much larger. This results from exten-
sive enthalpy/entropy compensation. The entropy
increases if the number of degrees of freedom in the
system increases, previously xed water molecules will
be released from the binding pocket into the bulk wa-
ter phase, or ahigh residual mobility will remain in
the binding pocket. This usually has adetrimental
effect on an increase in enthalpy, since the formation
of strong interactions is then hardly possible in an
efcient manner.
The pronounced interdependence of enthalpy and
-
entropy along with dynamic versus interaction–geo-
metric phenomena causes simple additive rules about
standardized functional group contributions to fail. In-
stead pronounced cooperative effects are in operation.
Bibliography and Further Reading
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Ajay and M. Murko, Computational Methods to Predict Binding
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I. D. Kuntz, K. Chen, K. A. Sharp and P.A. Kollman, The Maximal
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(1999)
J. E. Ladbury, Just add water! The effect of water on the specicity of
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′ Pocket of Thermolysin,
1