Tracing binding modes in hit-to-lead optimization: Chameleon-like poses of aspartic protease inhibitors

Article abstract of DOI:10.1002/anie.201411206

Successful lead optimization in structure-based drug discovery depends on the correct deduction and interpretation of the underlying structure-activity relationships (SAR) to facilitate efficient decision-making on the next candidates to be synthesized. Consequently, the question arises, how frequently a binding mode (re)-validation is required, to ensure not to be misled by invalid assumptions on the binding geometry. We present an example in which minor chemical modifications within one inhibitor series lead to surprisingly different binding modes. X-ray structure determination of eight inhibitors derived from one core scaffold resulted in four different binding modes in the aspartic protease endothiapepsin, a well-established surrogate for e.g. renin and β-secretase. In addition, we suggest an empirical metrics that might serve as an indicator during lead optimization to qualify compounds as candidates for structural revalidation.

Full text of DOI:10.1002/anie.201411206

Angewandte
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DOI: 10.1002/anie.201411206
Lead Structure Optimization
Tracing Binding Modes in Hit-to-Lead Optimization: Chameleon-Like
Poses of Aspartic Protease Inhibitors**
Maren Kuhnert, Helene Kçster, Ruben Bartholomꢀus, Ah Young Park, Amir Shahim,
Andreas Heine, Holger Steuber, Gerhard Klebe, and Wibke E. Diederich*
Abstract: Successful lead optimization in structure-based drug
discovery depends on the correct deduction and interpretation
of the underlying structure–activity relationships (SAR) to
facilitate efficient decision-making on the next candidates to be
synthesized. Consequently, the question arises, how frequently
a binding mode (re)-validation is required, to ensure not to be
misled by invalid assumptions on the binding geometry. We
present an example in which minor chemical modifications
within one inhibitor series lead to surprisingly different binding
modes. X-ray structure determination of eight inhibitors
derived from one core scaffold resulted in four different
binding modes in the aspartic protease endothiapepsin, a well-
established surrogate for e.g. renin and b-secretase. In addition,
we suggest an empirical metrics that might serve as an indicator
during lead optimization to qualify compounds as candidates
for structural revalidation.
make use of structure-educated decision-making in prioritiz-
ing medicinal chemistry activities.[2]
This optimization typically starts with one or more
identified hits sharing a common basic scaffold. The binding
mode of one representative with this scaffold and the target
protein is determined by crystallography, and subsequently
a series of derivatives is synthesized and their activity is
monitored by appropriate assays. Typically, structural modi-
fications are realized assuming that an identified binding
mode of a given parent scaffold is maintained during
structure–activity relationship (SAR) exploration and opti-
mization. However, despite the meanwhile long history of
structure-guided lead discovery there is an ongoing debate on
how frequently structural support is required to establish
a reliable SAR by monitoring putative changes of ligand
binding geometry. Moreover, what criteria can be applied to
indicate the requirement to assess putative conservation or
changes in the binding mode throughout the optimization
campaign? Herein, we follow these questions by utilizing
a well-suited model system and suggest advice that may
support the decision as to whether the (re)-validation of
a binding mode is required.
I
n the field of drug discovery and medicinal chemistry, the
concept of target-structure-based design considerations in the
optimization process from hit-to-lead and (pre-)clinical
candidate to an eventually approved drug has been well
established over the last three decades.[1] Success stories of
this structure-guided design approach range from the discov-
ery of various HIV-1 protease inhibitors via the design of the
neuraminidase inhibitor oseltamivir, the BCR-Abl kinase
inhibitor imatinib, and various HCV protease inhibitors to
a virtually unlimited number of current design campaigns that
In lead discovery, the aspartic protease endothiapepsin
(EP) commonly serves as a model enzyme and has success-
fully been exploited as a surrogate for renin and b-secretase in
structure-based inhibitor design.[3] Based on the Gewald
reaction (Scheme 1),[4] we have synthesized a series of
[*] M. Kuhnert, A. Shahim, Prof. Dr. W. E. Diederich
Institut fꢀr Pharmazeutische Chemie, Philipps-Universitꢁt Marburg
Hans-Meerwein-Strasse 3, 35032 Marburg (Germany)
E-mail: wibke.diederich@staff.uni-marburg.de
Scheme 1. General synthetic route to the 2-aminothiophene core
through the Gewald reaction.
Dr. H. Kçster, Dr. R. Bartholomꢁus, Dr. A. Y. Park, Prof. Dr. A. Heine,
Prof. Dr. G. Klebe
Institut fꢀr Pharmazeutische Chemie, Philipps-Universitꢁt Marburg
Marbacher Weg 6, 35032 Marburg (Germany)
Dr. H. Steuber[+]
substituted 2-aminothiophene-type EP inhibitors that cover
a broad range of affinities from two-digit mm up to submicro-
molar potency. Starting from the two-digit micromolar
inhibitors 1 and 2 (Figure 1) we embarked on a structure-
guided SAR exploration of this ligand series.
LOEWE-Zentrum fꢀr Synthetische Mikrobiologie, Philipps-Univer-
sitꢁt Marburg
Hans-Meerwein-Straße, 35032 Marburg (Germany)
[+] Current address: Bayer Pharma AG, Lead Discovery Berlin -
Structural Biology
The crystal structure of EP in complex with 1, determined
at 1.30 ꢀ resolution, unambiguously shows the ligandꢁs
tryptamine moiety bound into the S1 pocket, while the
thiophene core resides between the S1’ and S2 pockets
(Figure 2a). Key hydrogen bonds are formed between the
ligandꢁs sp3-hybridized nitrogen and the two aspartates of the
catalytic dyad (Asp35 and Asp219), mediated by the lytic
Mꢀllerstraße 178, 13353 Berlin (Germany)
[**] We acknowledge the support of the beamline staff at BESSY II,
Berlin (Germany), Petra III, Hamburg (Germany), and SLS,
Villingen (Switzerland) and travel grants from the Helmholtz-
Zentrum fꢀr Materialien und Energie, Berlin (Germany).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201411206.
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disordered prompted us to evaluate a more rigid scaffold
such as a tetrahydrobenzothiophene.
The synthesized compounds and their inhibitory activities
are shown in Figure 3, with 4 and 9 being the most potent
representatives (460 and 545 nm, respectively). However, the
interpretation of the underlying SAR turned out to be
challenging based on the initially determined binding geom-
etry of 1. Surprisingly, the simple ester-to-amide exchange
resulted in an affinity improvement by a factor of five (3,
4.0 mm). Is this gain solely attributed to the changed inter-
action of the introduced amide function, and
Figure 1. Chemical structures of 1 and 2 and their affinity data against
EP.
how does this relate to the intramolecular H-
bond observed in 1? The replacement of the
ethyl moiety in 3 by a benzyl substituent leads
to the most potent compound of our series, 4,
with a Ki value of 460 nm and thus an improve-
ment in the inhibitory activity of one order of
magnitude. Removal of the 4-phenyl substitu-
ent and annulation of a tetrahydrobenzene
moiety at the [b] site of the thiophene ring (6)
reduces affinity by about fourfold, although in
1 this substituent appeared disordered and was
not expected to contribute significantly to the
establishment of a directional interaction to EP.
The replacement of the indole moiety (4) by
a benzyl group (5), presumably accompanied by
the loss of the charge-assisted H-bond to Asp81,
Figure 2. a) Observed binding mode of 1 in complex with EP. Amino acids involved in
key hydrogen bonds (dashed lines) are shown as rods. For the detailed interaction
pattern, see Figure S5 in the Supporting Information. b) Sites for structural variations
derived from compound 1.
water molecule. Likely, this amine is protonated and serves as
a hydrogen-bond donor to the carbonyl group of Gly221. The
indole NH contributes a charge-assisted H-bond to Asp81
located at the tip of the hairpin-type flap region. Noteworthy,
for the 4-phenyl substituent of the ligand, no properly defined
difference electron density could be observed, suggesting
a distribution over multiple conformational states in the
binding site, and therefore it was omitted from the structural
model. Interestingly, the amide NH and the ester carbonyl
oxygen of the ligand establish an intramolecular H-bond,
keeping the ester and the thiophene plane approximately
coplanar (dihedral angle ca. 188) and the spatially demanding
substituents (ethyl ester and phenyl ring) in an eclipsed-type
conformation.
Based on the observed binding mode we designed and
synthesized a series of compounds with different structural
variations to explore the underlying SAR of this inhibitor
scaffold (Figure 2b). Via extension of the ethyl ester with
larger hydrophobic substituents aimed to address the hydro-
phobic S2/S4 pocket, a gain in affinity was anticipated. In
addition, to modulate the electronic properties of the intra-
molecular H-bond, the influence of an exchange of the ester
for an amide moiety was investigated. A thereby induced
change in the rotameric state of the carbonyl group might
additionally enable a polar contact to Tyr226 (Figure S1 in the
Supporting Information). Further sites for variation consisted
in changing the length of the tryptamine side chain as well as
the positioning of the basic nitrogen. The replacement of the
indole by morpholine was considered to enhance solubility.
Finally, the observation that the 4-phenyl substituent is
Figure 3. Chemical structures of the synthesized compounds 3–10 and
their corresponding Ki values against EP.
reduces the affinity by approximately twofold (1.1 mm). A
positional shift of the aliphatic nitrogen that was involved as
a donor in an H-bond to the lytic water resulted in 8 (1.5 mm)
and additionally a one-carbon insertion rendered 9, which,
with an activity of 545 nm, was unexpectedly found to be
nearly as potent as 4.
Overall, this rather incongruent SAR prompted us to
perform a comprehensive structural analysis of the underlying
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binding modes by crystal structure analysis. To our surprise,
the binding modes of the synthesized ligands successfully
determined for 1 and 3–9, split into four clusters of binding
poses, and our starting compound 1 turned out to represent
a structural singleton, in other words, the sole representative
of this binding mode. Figure 4a shows the crystal structures
determined in this study and the affiliation for each inhibitor
to the corresponding cluster, as well as the superposition of
one cluster representative with the binding pose of our initial
lead 1. Among the eight crystal structures, all adopting the
same crystal packing, the protein itself manifests nearly
identical conformation: rmsd values for the all-atom fitting
are below 0.5 ꢀ, backbone fitting yielded rmsd values below
0.3 ꢀ.[5] Hence, the deviating binding modes are neither
caused by significant rearrangements of the protein environ-
ment, nor are they induced by different crystal forms that
might create a different packing environment. Surprisingly,
the two most potent compounds belong to different binding
mode clusters. The largest cluster (green) comprises four
derivatives (3, 4, 5, and 6), followed by a two-membered
cluster (8 and 9) and two structural singletons (1 and 7).
Noteworthy, in between the four clusters, the binding
modes of the inhibitors with respect to the two catalytic
aspartates and the occupancy of the remaining binding
pockets differ substantially. The lytic water molecule adopts
a versatile role: it acts either as an H-bond donor or an H-
bond acceptor or is released from the complex. Inhibitor
1 addresses the Asp35/219 with its sp3-hybridized nitrogen via
the lytic water molecule, the latter acting as an H-bond
acceptor (Figure 4a). In the structures of 8 and 9 (purple
cluster) the benzyl-substituted amide nitrogen forms a polar
contact to the lytic water molecule, even though the
interaction geometry appears rather unfavorable for a typical
H-bond. Inhibitor 7 interacts through its morpholinomethy-
lene-substituted carbonyl oxygen mediated by the lytic water
molecule with the catalytic aspartates; in this case, however,
the water molecule acts as an H-bond donor. All representa-
tives of the green cluster directly address the catalytic dyad,
while the lytic water molecule is released upon binding of the
inhibitors. The significantly different binding modes provoke
substantially deviating H-bond networks between the inhib-
Figure 4. a) Left: Binding modes and assignment to binding mode clusters. Green, purple, yellow, and orange frames indicate the four different
binding modes. Resolutions of the X-ray structures are shown in ꢂ. The 2FoÀFc electron densities are depicted for the ligands as a blue mesh at
1s. Right: Superposition of 1 (yellow) with one representative of each binding mode cluster in the active site. b) H-bond of the amide NH of 3 to
Thr222 Og. Likely Thr222 forms an H-bond as an H-bond donor to Asp219 and therefore can only act as an H-bond acceptor to the ligand.
Distances in ꢂ. c) H-bond of the indole NH of 3, 4, and 6 to Asp81 and Ser83. This interaction is not possible for 5 due to the replacement of the
indole by benzyl. d) The charge-assisted H-bond interaction to Asp81, which in the green cluster (here shown for 4) is formed with the indole NH,
is established in 8 and 9 by means of the 2-amino function of the thiophene. e) H-bond network of 8 and 9 formed with the ligands’ aniline NH
or benzyl NH, respectively. Involved water molecules are shown in spheres.
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itors and the protein, which are shown in Figures S3–S6 in the
Supporting Information.
geometry, which enables 8 and 9 to maintain the charge-
assisted H-bond interaction to Asp81, although the indole
moiety that previously accomplished this task is replaced by
the phenyl/benzyl group with the amide NH in thiophene 2-
position (Figure 4d). Even though the aryl- and benzyl-
substituted nitrogens are located at virtually identical posi-
tions, the increase in affinity by about threefold from 8 to 9
might be explained by the different pKa values of the aniline
NH compared to the benzyl NH. Thus, the latter, which is
being most likely protonated to a larger extent, is capable of
forming a more efficient charge-assisted H-bond to the water
molecule that mediates the H-bond to Asp33 and Gly221
(Figure 4e).
In our inhibitor series also less or inactive compounds
were synthesized which are essential for SAR interpretations
as well. However, in the present case the interpretation of
these data is almost impossible, as the assignment to one of
the binding mode clusters remains speculative.
The current example highlights the complexity of binding
events and their strong dependence on seemingly minor
effects of scaffold decoration and modifications. That 1, 7, 8,
and 9 do not adopt the same binding geometry as observed in
the green cluster can be rationalized retrospectively, but
without the crystal structure determinations the adopted
binding modes would have been difficult to predict, and, if so,
would have hardly been believed or attracted sustained
attention without our experimental evidence.
Such an unexpected flipping of binding modes after
modification of the scaffold substitutions has been detected
sporadically, however, mostly by serendipity.[6] As the com-
monly accepted hypothesis that similar ligands bind in
a similar fashion[7] apparently does not hold true on a com-
prehensive basis, the question arises, how frequently a
(re-)validation of an assumed binding mode is required. Are
there any easily available indicative hints to estimate whether
a crystallographic (re-)validation of the assumed binding
mode used as working hypothesis is required throughout the
lead exploration campaign?
In addition to the affinity determination via a fluores-
cence-based assay (Ki values), we performed a thermal shift
assay (TSA) to investigate the extent to which the studied
compounds stabilize the protein, especially with respect to the
different binding modes. TSAs are commonly used for hit
identification and affinity ranking of inhibitors.[8,9]
The results for our inhibitor series (Table 1) clearly
suggest that the observed shifts in the melting temperature
do not generally correlate with the Ki values. However, within
one binding mode cluster, the DTm values are ranked
correctly, with 5 being an exception: This might be attributed
to the loss of the charge-assisted H-bond to Asp81 due to the
absence of the indole moiety, while the other members of this
cluster establish this interaction. This observation is in
agreement with the concept that TSA measurements require
similar binding enthalpies for correct affinity ranking.[9]
However, how do TSA data perform for ligands of identical
chemotype that fall in the same affinity range, but exhibit
different binding geometries?
The binding mode of 1 apparently relies on the presence
of the ester moiety, whereas the amide derivative (3) adopts
an altered binding mode. Interestingly, the intramolecular H-
bond observed in 1 (Figure 2a) breaks up in favor of the
establishment of an H-bond to Thr222 Og through the ethyl-
substituted amide nitrogen. How does the mere replacement
of the ester by an amide translate into such a predominant
change in binding geometry? In all the EP crystal structures
determined in our study, the interaction pattern between
Thr222 Og and the Asp219 carboxylate (2.7 ꢀ) is conserved .
Likely, this Thr side chain donates an H-bond to the
carboxylate of Asp219 keeping the proton of Thr222 Og
oriented towards the aspartate (Figure 4b). This observation
supports the conclusion that Thr222 Og itself can only be
involved in favorable polar interactions if addressed by
appropriate H-bond donors on the ligand site. This is
impossible for the ester 1 as its oxygen can only serve as an
H-bond acceptor; hence the ester prefers to saturate its polar
contact inventory by an intramolecular H-bond. However,
when the ester is changed to an amide (3), the introduced NH
function is able to address Thr222 Og as a donor rather than
maintaining the intramolecular H-bond, while the concom-
itantly nonsaturated glycine amide NH rotates by ꢀ 1008 to
establish an H-bond to the Asp219 carboxylate (Figure 4a).
Although the overall SAR interpretation is inconclusive
in the absence of structural data, the SAR within the clusters
reveals a consistent picture, which will be discussed only
exemplarily. In the green cluster, 3 bearing an ethyl group at
P2 has the lowest affinity (4.0 mm); this most likely results from
the reduced hydrophobic interactions, compared to those of
the inhibitors equipped with a benzyl group at this position.
Furthermore, 3 and 6 lack the intramolecular p–p interaction
between the two phenyl rings observed in 4 and 5, which leads
to a loss of preorganization corresponding to the bound
conformation. In 5, due to the exchange of the indole moiety
for the benzyl group, the H-bond to Asp81 and the weak polar
contact to Ser83 as observed in 3, 4, and 6 cannot be
established (Figure 4c). In 4, the representative with the
highest affinity (460 nm) of this cluster, the four varied
substituents seem to be optimally chosen among all inves-
tigated variations, as the indole moiety is able to form an H-
bond to Asp81 whereas the benzyl moiety interacts via p–p
stacking to Tyr226. Inhibitor 8 (purple cluster) differs from 5
(green cluster) only by the position of the secondary amino
nitrogen in the P1 substituent of the inhibitor. Based on the X-
ray structure of 5, a lower affinity caused by a less efficient
interaction of the relocated nitrogen to the catalytic aspar-
tates would have been expected for 8. Interestingly though, its
affinity is nearly unchanged. The X-ray structure of 8 provides
the explanation for this initially surprising observation: The
switch in binding geometry between 5 and 8 seems to be
mainly triggered by the loss of the H-bond to Asp35 that
would be expected for an unchanged geometry of 5. The
thiophene moiety formerly located in the S1’ pocket (green
cluster) flips by about 1808 in order to populate now the S2
pocket. Likewise, inhibitor 9, in which the P1 substituent is
extended by one carbon atom, shows this altered binding
Only two of our four binding mode clusters qualify for
such a comparison, as the singletons 1 and 7 are slightly off-
4
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Table 1: TSA data ordered by decreasing DTm values, as well as
assignment to the binding mode clusters (color coded) and Ki values.
be rather meaningless and will likely drive scaffold optimi-
zation in the wrong direction. We expect that our case study
and the application of the suggested validation protocol will
encourage drug designers to further pursue and study chemi-
cally attractive scaffolds albeit an at first glance seemingly
inconclusive SAR. This is likely to be rewarded with a more
comprehensive understanding and an increasing number of
starting options for further lead optimization.
Experimental Section
Protein Databank codes for this manuscript: 1 (3T7Q), 3 (4L6B), 4
(3PSY), 5 (3WZ6), 6 (3WZ7), 7 (3T7X), 8 (3WZ8), 9 (4LAP).
Received: November 19, 2014
Published online: && &&, &&&&
Keywords: drug design · enzyme inhibitors ·
.
ligand–enzyme binding · structure determination ·
thermal shift assay (TSA)
range with respect to their affinity. However, the affinities of
the green and the purple clusters cover a similar affinity range
thus permitting such a comparison. Interestingly, for these
two clusters, ligands of rather identical affinity deviate with
respect to their associated TSA profile: The two most potent
ligands 4 and 9 possess affinities of 460 nm and 545 nm,
respectively, but differ by 0.88C in their TSA profiles (4.6
versus 3.88C) which corresponds to a deviation of more than
threefold of the standard deviation of the measurement.
Analogously, 5 and 6 inhibit EP with a virtually identical
affinity of 1.1 and 1.8 mm, respectively, and 8 is equally potent
(1.5 mm). However, the latter exhibits a TSA shift of only
0.48C, while the first two ligands significantly enhance the
stability of the protein as indicated by a shift of DTm of 2.2 and
3.28C, respectively (with 5 being the abovementioned internal
outlier within the green cluster). These observations suggest
that protein stabilization by a particular ligand depends not
only on affinity, but also on its adopted interaction pattern.
This is also true for the comparison of 1 and 8, both of which
exhibit a similar TSA shift (0.7 vs. 0.48C, respectively) but
possess a substantial difference in affinity (18 vs. 1.5 mm), thus
suggesting that they stabilize the target protein by deviating
interaction geometries.
Hence, discrepancies between affinity data based on
enzyme kinetics and TSA results within one inhibitor series
may be exploited as a hint for putative changes in the adopted
binding geometry, which may qualify the seeked “outlier”-
type ligands as promising candidates for revalidation of the
assumed binding mode.
The presented example impressively points out that minor
structural changes within one lead series, such as the replace-
ment of an ester by an amide or the introduction of an
additional carbon atom, may result in entirely different
binding modes. An inconsistent affinity–TSA correlation may
serve as an easily available indicator to qualify candidates for
structural revalidation, even if the interaction mode is
believed to be known. In the absence of such structural
data, a reasonable interpretation of substituent effects or
assumed bioisosteric replacements at a given chemotype will
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Communications
Lead Structure Optimization
M. Kuhnert, H. Kçster, R. Bartholomꢁus,
A. Y. Park, A. Shahim, A. Heine,
H. Steuber, G. Klebe,
W. E. Diederich*
&&& — &&&
Tracing Binding Modes in Hit-to-Lead
Optimization: Chameleon-Like Poses of
Aspartic Protease Inhibitors
Changing poses: The optimization of lead
structures relies on the systematic varia-
tion of the substituents decorating
a given hit scaffold which is based on one
binding pose that is supposed to be
invariable. For a given core scaffold, only
minor chemical variations were found to
result in four different binding modes. An
indicative metrics is suggested to assess
candidates that qualify for structural
revalidation.
6
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