Structure-based design of inhibitors of the aspartic protease endothiapepsin by exploiting dynamic combinatorial chemistry

Article abstract of DOI:10.1002/anie.201309682

Structure-based design (SBD) can be used for the design and/or optimization of new inhibitors for a biological target. Whereas de novo SBD is rarely used, most reports on SBD are dealing with the optimization of an initial hit. Dynamic combinatorial chemistry (DCC) has emerged as a powerful strategy to identify bioactive ligands given that it enables the target to direct the synthesis of its strongest binder. We have designed a library of potential inhibitors (acylhydrazones) generated from five aldehydes and five hydrazides and used DCC to identify the best binder(s). After addition of the aspartic protease endothiapepsin, we characterized the protein-bound library member(s) by saturation-transfer difference NMR spectroscopy. Cocrystallization experiments validated the predicted binding mode of the two most potent inhibitors, thus demonstrating that the combination of de novo SBD and DCC constitutes an efficient starting point for hit identification and optimization. The dynamic duo: The combination of de novo structure-based design and dynamic combinatorial chemistry has been applied to the identification of novel acylhydrazone-based inhibitors for the aspartic protease endothiapepsin. ¹H-STD-NMR spectroscopy has been used to identify the binders from the dynamic combinatorial libraries. Proposed binding modes of the most potent inhibitors have been confirmed by X-ray crystallography.

Full text of DOI:10.1002/anie.201309682

Angewandte
Chemie
DOI: 10.1002/anie.201309682
Enzyme Inhibitors
Structure-Based Design of Inhibitors of the Aspartic Protease
Endothiapepsin by Exploiting Dynamic Combinatorial Chemistry**
Milon Mondal, Nedyalka Radeva, Helene Kçster, Ahyoung Park, Constantinos Potamitis,
Maria Zervou, Gerhard Klebe,* and Anna K. H. Hirsch*
Abstract: Structure-based design (SBD) can be used for the
design and/or optimization of new inhibitors for a biological
target. Whereas de novo SBD is rarely used, most reports on
SBD are dealing with the optimization of an initial hit.
Dynamic combinatorial chemistry (DCC) has emerged as
a powerful strategy to identify bioactive ligands given that it
enables the target to direct the synthesis of its strongest binder.
We have designed a library of potential inhibitors (acylhydra-
zones) generated from five aldehydes and five hydrazides and
used DCC to identify the best binder(s). After addition of the
aspartic protease endothiapepsin, we characterized the protein-
bound library member(s) by saturation-transfer difference
NMR spectroscopy. Cocrystallization experiments validated
the predicted binding mode of the two most potent inhibitors,
thus demonstrating that the combination of de novo SBD and
DCC constitutes an efficient starting point for hit identification
and optimization.
from the DCL. Saturation-transfer difference (STD) NMR
spectroscopy, which enables the direct characterization of
target—ligand interactions in solution,[3] has been applied to
identify DCL members bound to the target.[4] In addition to
DCC, structure-based design (SBD) is also a powerful
strategy to design and/or optimize bioactive compounds.[5]
Whereas de novo SBD is rarely used,[6] most reports on SBD
deal with the optimization of an initial hit discovered by other
means. Therefore, the combination of SBD and DCC would
represent a highly efficient hit-identification strategy in
a range of medicinal-chemistry projects. In the present
study, we have used de novo SBD in combination with
1
acylhydrazone-based DCC and H-STD-NMR spectroscopy
to identify a new family of potent hits for endothiapepsin,
which belongs to the notoriously challenging class of aspartic
proteases.[7] Finally, we have validated the proposed binding
mode of the inhibitors by X-ray crystallography.
Endothiapepsin belongs to the family of pepsin-like
aspartic proteases, which are involved in a wide range of
diseases such as hypertension and malaria.[7] Endothiapepsin
has been used as a model enzyme for mechanistic studies[8] as
well as for the development of renin[9] and b-secretase[10]
inhibitors. Eukaryotic aspartic proteases comprise two struc-
turally similar subunits, each contributing an aspartic acid
residue to the catalytic dyad (Asp35 and Asp219 in endo-
thiapepsin) that cleaves the peptide bond of the substrate
using a bound water molecule.
O
ver the past decade, dynamic combinatorial chemistry
(DCC)[1] has emerged as a powerful strategy to identify
ligands for biological targets.[2] DCC allows the formation of
a dynamic combinatorial library (DCL), in which the bonds
between the building blocks are reversible. Upon addition of
a target, one or more library members are bound, thereby
leading to selection and amplification of the strongest binders
[*] M. Mondal, Dr. A. K. H. Hirsch
The formation of an imine-type bond has been applied to
medicinal-chemistry-based DCC projects.[11] In aqueous so-
lution, which is required for any biological application, imines
themselves are inherently too unstable. Acylhydrazones, on
the other hand, offer the right kinetic and thermodynamic
balance.[12] Despite its pH dependence, this type of reversible
linkage has started to attract attention.
This linkage requires the target protein to be stable at
room temperature for one week (pH 7.2),[2c] the use of aniline
as a nucleophilic catalyst (pH 6.2),[13] or an acidic buffer
system (pH < 6). Acylhydrazones are attractive for biological
DCC given that the building blocks are readily available,
afford an amide-type linkage that offers both hydrogen-bond
donor and -acceptor sites for molecular recognition by the
target and are sufficiently stable both at acidic and physio-
logical pH values (see Figure S1 in the Supporting Informa-
tion) to enable direct analysis of the DCL. The optimum
pH value of endothiapepsin is 4.5, and we have shown that the
enzyme is stable under these conditions at room temperature
for more than 20 days (see Figure S2 in the Supporting
Information), thus making it an ideal target enzyme for an
SBD project that exploits acylhydrazone-based DCC.
Stratingh Institute for Chemistry, University of Groningen
Nijenborgh 7, 9747 AG Groningen (The Netherlands)
E-mail: A.K.H.Hirsch@rug.nl
Homepage: http://www.rug.nl/research/bio-organic-chemistry/
hirsch/
N. Radeva, Dr. H. Kçster, Dr. A. Park, Prof. Dr. G. Klebe
Institute of Pharmaceutical Chemistry
Marbacher Weg 6, 35032 Marburg (Germany)
E-mail: Klebe@Staff.Uni-Marburg.de
Dr. C. Potamitis, Dr. M. Zervou
Institute of Biology, Medicinal Chemistry & Biotechnology
National Hellenic Research Foundation
48,Vas. Constantinou Ave, Athens 11635 (Greece)
[**] We thank Pieter van der Meulen for useful suggestions and
discussions about STD NMR spectroscopy. Funding was granted by
the Netherlands Organisation for Scientific Research (NWO-CW,
VENI grant to A.K.H.H.), by the Dutch Ministry of Education,
Culture, Science (Gravity program 024.001.035) and European
Union’s Seventh Framework Programme (FP7-REGPOT-2009-1)
under grant agreement no. 245866 (to C.P. and M.Z.), and by the
ERC advanced grant no. 268145 DrugProfilBind kindly provided by
the EU.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201309682.
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We have used two crystal structures of endothiapepsin
(PDB: 3PBD and 3PI0) that represent two alternative
binding modes: with and without a crystallographically local-
ized water molecule. We did not use the fragments as
a starting point but only the structure of the enzyme.[14] We
designed a series of acylhydrazones to address the catalytic
dyad through hydrogen-bonding interactions, by using the
molecular-modeling software MOLOC[15] and the FlexX
docking module in the LeadIT suite.[16] The acylhydrazone
moiety appeared to be a suitable central scaffold, as modeling
suggested it was anchored to the active site through a strong
hydrogen-bond network with the catalytic dyad. We intro-
duced an a-amino group that can form charge-assisted
hydrogen bonds to the catalytic dyad as well as to Asp81,
Asp33, Gly221, or Thr222. We modeled two alternative
hydrazone-based inhibitors (see Figure S3 and Table S1 in the
Supporting Information for selected docking results), retro-
synthesis of which led to five hydrazides H1–H5 and five
aldehydes A1–A5 (Scheme 1).
Whereas all the aldehydes as well as hydrazide H5 are
commercially available, hydrazides H1–H4 were obtained in
80–90% yield from their corresponding enantiomerically
pure methyl esters by treatment with hydrazine monohydrate
(Scheme S1 in the Supporting Information). For the synthesis
of hydrazide H2, we took advantage of an asymmetric
Strecker reaction[18] starting from commercially available
para-fluorobenzaldehyde (see Scheme S2 in the Supporting
Information).
1
We used H-STD-NMR experiments to identify the best
component associations bound to endothiapepsin, as this
approach enables bound ligands to be monitored and requires
only small amounts of unlabeled protein.[3] To facilitate the
analysis, we divided the whole library into five sublibraries,
each consisting of five hydrazides and one aldehyde, thus
resulting in the formation of five potential acylhydrazones
(ten isomers, including E/Z isomers) in equilibrium with the
initial building blocks (see Scheme S3 in the Supporting
Information). The potential formation of imine products with
the free amino group of the hydrazides H1–H4 or a surface-
exposed Lys side chain can be ruled out because of the use of
acidic conditions.[19] After addition of the target enzyme to the
pre-equilibrated libraries, we identified bound acylhydra-
zones by analyzing the imine-type (see Figures S4, S5, S6, and
S7 in the Supporting Information) and a-carbon (see Fig-
ure S8 in the Supporting Information) proton signals of
Figure 1. Superposition of MOLOC-generated molecular models of
potential inhibitors featuring a) direct hydrogen bonding with the
catalytic dyad in the active site of endothiapepsin (PDB code: 3PBD)
and b) hydrogen bonding with the catalytic dyad in the active site of
endothiapepsin through the catalytic water molecule (PDB code:
3PI0). Color code: protein backbone: gray; inhibitor skeletons: C:
green, violet, purple, blue; N: dark blue; F: cyan; O: red; crystallo-
graphically localized water molecule: red sphere.
1
acylhydrazones in the H-STD-NMR spectra. We identified
a total of eight binders from the five sublibraries (Scheme 1).
binding poses, in which the acylhy-
drazone addresses the catalytic dyad
either directly or via the catalytic
water molecule (Figure 1). During
the modeling study, the E isomers
emerged as the most suitable scaf-
folds, displaying two vectors pointing
towards the S2, S1, S1’, and S2’
pockets. Inspection of the cocrystal
structures of endothiapepsin with 11
fragments,[14] as well as hot-spot[17]
analyses of the active site of endo-
thiapepsin showed that both aro-
matic and aliphatic substituents can
be accommodated in the S2, S1, S1’,
and S2’ pockets surrounding the
catalytic dyad. According to our
modeling study, both mono- and
bicyclic aromatic moieties can be
accommodated by the S1’/S2’
pocket, whereas the hydrophobic S2
pocket is best occupied by a mesityl
substituent. The S1/S1’ pocket can
host an indolyl, isobutyl, or phenyl
Scheme 1. Dynamic formation of an acylhydrazone library and enzymatic selection of the best
moiety. We selected a series of acyl- inhibitors by 1H-STD-NMR analysis.
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To differentiate specific from nonspecific binding, we added
the potent inhibitor saquinavir (Ki = 48 nm) to the H1–5 + A4
sublibrary, thereby leading to the appearance of its signals at
gies of binding (DG) and ligand efficiencies (LEs), obtained
from the IC50 values using the Cheng–Prusoff Equation,[21]
correlate with the calculated values from the scoring function
HYDE in the LeadIT suite (DGHYDE((S)-H4-A4) =
1
the expense of the acylhydrazones signals in the H-STD-
NMR spectrum, thus confirming specific binding of the
acylhydrazones H3-A4 and H4-A4 (see Figure S9 in the
Supporting Information).
À32 kJmolÀ1, DGHYDE ((R)-H3-A5) = À26 kJmolÀ1
;
see
Table 1 and Table S1 in the Supporting Information).[22]
To validate the predicted binding mode from SBD, we
soaked crystals of endothiapepsin with the two most potent
inhibitors and were able to determine crystal structures of
(R)-H3-A5 (PDB code: 3T7P) and (S)-H4-A4 (PDB code:
4KUP) in complex with endothiapepsin at 1.25 ꢀ and 1.31 ꢀ
resolution, respectively (see Figures S16—S19 in the Support-
ing Information). During the soaking experiments, the
enzyme selects the E isomer from the mixture of E/Z isomers.
The cocrystal structure of (R,E)-H3-A5 shows two ligands
in the active site: one binds to the catalytic dyad, whereas the
second is oriented towards the solvent. As a result of solvent
exposure and lack of protein contacts, the portion of this
ligand molecule not visible in the electron density map is most
likely highly mobile and scattered over various conforma-
tional states. The (S,E)-H4-A4 complex also exhibits two
ligand molecules in the binding pocket. Of these, only the first
binds to the catalytic dyad, whereas the second ligand
molecule is solvent-exposed and, by analogy to the previous
case, only fractionally visible in the electron density and
partially occupied (63%; Figure 2).
To investigate the biological activity of the hits identified
by H-STD-NMR, we performed inhibition studies using the
1
acylhydrazones (S)-H1-A1, (S)-H1-A3, (S)-H2-A1, (R)-H3-
A1, (R)-H3-A4, (R)-H3-A5, (S)-H4-A4, and H5-A1 synthe-
sized from their corresponding aldehyde and hydrazide
precursors (see Schemes S4 and S5 in the Supporting
Information). We determined their inhibitory potency using
the mixture of E/Z isomers by applying a fluorescence-based
assay adapted from the HIV-protease assay (see Fig-
ures S10—S15 in the Supporting Information).[20]
The enzyme-inhibition assay confirmed the result of the
1H STD NMR experiments. All eight acylhydrazones indeed
inhibit endothiapepsin with IC50 values in the range of 13 to
365 mm. The most potent inhibitors, acylhydrazones (S)-H4-
A4 and (R)-H3-A5, feature IC50 values of 12.8 mm and
14.5 mm, respectively (Table 1). The experimental free ener-
Table 1: IC50 values, calculated Ki values, DG values, and LEs of eight
acylhydrazones identified as inhibitors of endothiapepsin.[a]
Inhibitor
IC50 [mm]
Ki [mm]
DG [kJmolÀ1
]
LE
To clarify whether the additional ligands observed are
only present because of the high ligand concentrations
applied during the soaking, we prepared crystals at different
concentrations (2 mm, 20 mm, and 100 mm) and collected
separate datasets (PDB codes: 4LHH, 4KUP, and 4LBT).
Remarkably, at the lowest concentration, only the binding
mode engaging the catalytic dyad is observed. At the highest
concentration, population of a third binding pose is apparent
(see Figure S20 in the Supporting Information). We therefore
believe that only the binding poses of (R,E)-H3-A5 and (S,E)-
H4-A4 next to the catalytic dyad are relevant for our
considerations.
(S)-H4-A4
(R)-H3-A5
(S)-H1-A1
(S)-H2-A1
(R)-H3-A4
(R)-H3-A1
(S)-H1-A3
H5-A1
12.8Æ0.4
14.5Æ0.5
150Æ17
177Æ13
206Æ19
352Æ13
365Æ95
insoluble
6Æ0.2
7Æ0.2
71Æ8
À30
À30
À24
À23
À23
À22
À22
–
0.27
0.29
0.27
0.23
0.25
0.22
0.30
–
83Æ6
97Æ9
166Æ6
172Æ45
[a] Values indicate the inhibition constant (Ki), the Gibbs free energy of
binding (DG), and ligand efficiency (LE) derived from the Cheng–Prusoff
Equation.[21] Full details of the biological assay conditions are provided in
the Supporting Information.
Figure 2. X-ray crystal structures of endothiapepsin cocrystallized with ligands: a) Overview of the two molecules of (R,E)-H3-A5 (C: green, water
molecules: green spheres) bound in the active site (PDB code: 3T7P). The central ligand binds through the catalytic water molecule to Asp35 and
Asp219. b) Overview of both molecules of (S,E)-H4-A4 (C: yellow, water molecule: yellow sphere; PDB code: 4KUP). The central ligand addresses
Asp35 and Asp219 directly through its a-amino group. c) Superposition of (R,E)-H3-A5 and (S,E)-H4-A4. Only the surface of the (R,E)-H3-A5
complex is shown.
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The detailed binding modes of both ligands are shown in
Figure 2 and Figures S11 and S12 in the Supporting Informa-
tion. The amide NH group of (R,E)-H3-A5 forms a hydrogen
bond to the dyad, mediated by the catalytic water molecule
(Figure 2a). The imine-type N atom of the acylhydrazone
linker forms a hydrogen bond with the backbone amide NH
group of Gly80 (2.9 ꢀ). Another hydrogen bond is observed
between the carbonyl group of the acylhydrazone and the
backbone amide NH group of Asp81 (3.0 ꢀ). The hydroxy
group of the naphtholyl portion of the second ligand donates
a hydrogen bond via its hydrogen atom to the outer O atom of
the side chain of Asp219 (2.8 ꢀ). Furthermore, the a-amino
group of the ligand forms a weak hydrogen bond to an
adjacent water molecule, with the remainder of the ligand
being bound in the S1’ and S2 pockets and oriented towards
the solvent.
(S,E)-H4-A4 interacts differently with the catalytic dyad
(Figure 2b) as it uses its a-amino group to form direct
hydrogen bonds to Asp35 (2.8 ꢀ, 3.2 ꢀ) and Asp219 (2.9 ꢀ);
the a-amino group occupies virtually the same position as the
catalytic water molecule in the complex with (R,E)-H3-A5.
While the amide-type NH group of the acylhydrazone linker
forms a hydrogen bond to the hydroxy group of the Thr222
side chain (2.8 ꢀ), the indolic NH group donates its proton to
form a hydrogen bond to the carboxylate group of Asp81
(3.0 ꢀ).
(R,E)-H3-A5 and (S,E)-H4-A4 occupy different regions
of the binding pocket of endothiapepsin. Clearly, the different
binding poses are provoked by the inverted stereochemistry
at Ca. This allows (S,E)-H4-A4 to address the S1 pocket with
its indolyl moiety benefiting from CH–p interactions with
Phe116 and Leu125 and to form a salt bridge with the catalytic
dyad. Furthermore, (R,E)-H3-A5 places its hydrophobic
phenyl group in the S1 pocket and is engaged in CH–p and
p–p interactions with Leu125 and Phe116, but its stereo-
chemistry does not allow an interaction with the dyad. Instead
the water-mediated contact through its acylhydrazone linker
is achieved. This ligand benefits from CH–p interactions with
Ile77 and Leu133 through its naphtholyl moiety in the S2’
pocket, whereas (S,E)-H4-A4 experiences some hydrophobic
contacts with Ile300 and Ile304 in the S2 pocket. The binding
poses indicated by the additional ligand molecules soaked
into the crystals at higher concentrations map out secondary
interaction sites most likely experiencing minor affinity
contributions.
In conclusion, we have demonstrated for the first time that
the combination of de novo SBD and DCC is a powerful
technique for the rapid identification of novel hits that inhibit
the aspartic protease endothiapepsin. We have exploited 1H-
STD-NMR spectroscopy to identify the binders directly from
the DCL. Among the hits identified, the best ones exhibit
IC50 values in the low micromolar range. Subsequent cocrys-
tal-structure determination confirmed our in silico prediction
that either direct or water-mediated interactions with the
catalytic dyad can be achieved. We have reported the first
example of acylhydrazone-based inhibitors of endothiapepsin
and aspartic proteases in general. Our synergistic combina-
tion of methods holds great promise for the acceleration of
the drug-discovery process, not only for the notoriously
challenging class of aspartic proteases but for a wide range of
drug targets. By enabling the identification and concomitant
optimization of novel inhibitors, its potential would appear to
be greatest when applied during the early stages of the drug-
discovery process.
Received: November 6, 2013
Published online: February 14, 2014
Keywords: dynamic combinatorial chemistry ·
.
enzyme inhibitors · STD NMR spectroscopy ·
structure-based design · X-ray diffraction
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