Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment-Based Drug Design Facilitated by Dynamic Combinatorial Chemistry

Article abstract of DOI:10.1002/anie.201603074

Fragment-based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit-identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X-ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis-acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC₅₀value of 54 nm, which represents a 240-fold improvement in potency compared to the parent hits. Subsequent X-ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit-identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit-to-lead optimization.

Full text of DOI:10.1002/anie.201603074

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201603074
German Edition: DOI: 10.1002/ange.201603074
Drug Design
Fragment Linking and Optimization of Inhibitors of the Aspartic
Protease Endothiapepsin: Fragment-Based Drug Design Facilitated by
Dynamic Combinatorial Chemistry
Milon Mondal, Nedyalka Radeva, Hugo Fanlo-Virgꢀs, Sijbren Otto, Gerhard Klebe, and
Anna K. H. Hirsch*
[
6,7]
Abstract: Fragment-based drug design (FBDD) affords active
compounds for biological targets. While there are numerous
reports on FBDD by fragment growing/optimization, fragment
linking has rarely been reported. Dynamic combinatorial
chemistry (DCC) has become a powerful hit-identification
strategy for biological targets. We report the synergistic
combination of fragment linking and DCC to identify inhib-
itors of the aspartic protease endothiapepsin. Based on X-ray
crystal structures of endothiapepsin in complex with fragments,
we designed a library of bis-acylhydrazones and used DCC to
identify potent inhibitors. The most potent inhibitor exhibits an
IC50 value of 54 nm, which represents a 240-fold improvement
in potency compared to the parent hits. Subsequent X-ray
crystallography validated the predicted binding mode, thus
demonstrating the efficiency of the combination of fragment
linking and DCC as a hit-identification strategy. This approach
could be applied to a range of biological targets, and holds the
potential to facilitate hit-to-lead optimization.
become the favorite optimization strategy,
even though it
involves cycles of iterative design, synthesis and validation of
the binding mode of each derivative. To overcome this
drawback, we have previously reported the combination of
fragment growing and dynamic combinatorial chemistry
(DCC) to accelerate drug discovery. Fragment linking, on
the other hand, is attractive because of the potential for super-
additivity (an improvement in ligand efficiency (LE) rather
than mere maintenance of LE). The first example of fragment
linking was reported by Fesik and co-workers.
a few studies demonstrating the efficiency of fragment linking
of low-affinity fragments to produce higher-affinity ligands
The challenge lies in preserving the
binding modes of the fragments in adjacent pockets whilst
[8]
[
4,9]
Since then,
[
10,11]
have been reported.
[
12,13]
identifying a linker featuring an optimal fit.
[
14–18]
In addition to FBDD, DCC
screening (DLS)
and dynamic ligation
are powerful strategies for identifying/
[
19–22]
optimizing hit compounds for biological targets. In a dynamic
combinatorial library (DCL), the bonds between the building
blocks are reversible and are continuously being made and
broken. Addition of the target protein leads to re-equilibra-
tion as one or more library components are bound to the
protein, resulting in amplification of the strongest binder(s)
from the DCL. In DLS, formation of a reversible covalent
bond between a directing probe and a nucleophilic fragment
enables the detection of low-affinity ligands while measuring
at micromolar concentrations.
We therefore envisaged the potentially synergistic combi-
nation of fragment linking and DCC as an efficient hit-
identification/optimization strategy. In this work, we com-
bined fragment linking and bis-acylhydrazone-based DCC to
identify inhibitors for endothiapepsin, which belongs to the
notoriously challenging family of pepsin-like aspartic pro-
O
ver the past decade, fragment-based drug design (FBDD)
has emerged as a novel paradigm in drug discovery and it has
[1–3]
been applied to a growing number of biological targets.
FBDD has higher hit rates than high-throughput screening
and enables coverage of the chemical space using smaller
[
2]
[4]
libraries. Since its inception in the mid-1990s, FBDD has
expanded tremendously and various pharmaceutical compa-
nies have used FBDD to develop more than 18 drug
[
5]
candidates that are now in clinical trials.
After the identification of fragment hits by various
screening techniques, the hits are optimized to lead com-
pounds and drug candidates by fragment growing, linking,
and/or merging. Fragment growing, on the one hand, has
[
23]
teases.
[*] Dr. M. Mondal, Prof. Dr. A. K. H. Hirsch
Aspartic proteases are found in fungi, vertebrates, plants,
and retroviruses such as HIV. This class of enzymes play
a causative role in important diseases such as malaria,
Alzheimerꢀs disease, hypertension, and AIDS. Owing to
its high similarity with these drug targets, endothiapepsin has
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/chemical-biology/hirsch/
[23]
N. Radeva, Prof. Dr. G. Klebe
Institute of Pharmaceutical Chemistry
Marbach Weg 6, 35032 Marburg (Germany)
[24–26]
been used as a model enzyme for mechanistic studies
and
[27]
[28]
for the discovery of inhibitors of renin and b-secretase.
Endothiapepsin is a robust enzyme, which remains active for
more than 20 days at room temperature, is readily available in
large quantities, and crystallizes easily, thus making it a useful
representative for aspartic proteases. Pepsin-like aspartic
proteases are active as monomers and consist of two
structurally similar domains, each of which donates an
Dr. H. Fanlo-Virgꢀs, Prof. Dr. S. Otto
Centre for Systems Chemistry, Stratingh Institute for Chemistry,
University of Groningen
Nijenborgh 4, 9747 AG Groningen (The Netherlands)
[
18]
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under http://dx.doi.org/10.
1002/anie.201603074.
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aspartic acid residue to the catalytic dyad (D35 and D219 in
endothiapepsin), which hydrolyzes the peptide bond of the
substrate through nucleophilic attack by a catalytic water
molecule.
Although bis-acylhydrazone-based DCC has been
[
29]
reported, there are no reports of fragment linking using
DCC. Herein, we describe the combination of fragment
linking/optimization and DCC to efficiently afford ligands for
inhibiting the aspartic protease endothiapepsin.
We chose X-ray crystal structures of endothiapepsin in
complex with acylhydrazones 1 and 2 (PDB IDs: 4KUP and
3
T7P, respectively) as a starting point for fragment linking
(
Figure 1). We had previously identified 1 and 2 as hits from
Figure 1. Structures of hits 1 and 2.
Figure 2. a) Superimposition of the crystallographically determined
binding modes of 1 (C: orange) and 2 (C: green) (PDB IDs: 4KUP and
3
T7P, respectively) with a putative bis-acylhydrazone inhibitor (C:
an acylhydrazone-based DCL using the synergistic combina-
tion of de novo SBDD and DCC. Our hits 1 and 2 displayed
IC50 values of 12.8 mm and 14.5 mm and ligand efficiencies
[
18]
yellow). b) Chemical structure of the modeled bis-acylhydrazone
shown in Figure 2a.
[33]
(
LEs) of 0.27 and 0.29, respectively, against endothiapepsin.
Both hits displayed alternative binding modes with the
catalytic dyad: either through a water-mediated interaction
or through direct interaction, with displacement of the lytic
water molecule. Fragments 1 and 2 occupy the S1 and S2 or S1
and S2’ pockets, respectively (Figure 2a).
Retrosynthetic analysis of the designed bis-acylhydra-
zones led to isophthalaldehyde (3) and nine hydrazide
building blocks (4–12) for DCC (Scheme 1). Whereas the
bis-aldehyde 3 and hydrazides 10–12 are commercially
available, we synthesized hydrazides 4–9 from their corre-
sponding methyl esters through treatment with hydrazine
monohydrate (60–90% yield, see Scheme S1 in the Support-
ing Information).
We set up a DCL with bis-aldehyde 3 and the nine
hydrazides 4–12, which has the potential to produce 78 bis-
acylhydrazones (excluding E/Z isomers) and 12 mono-acyl-
hydrazones. To facilitate the analysis, we divided the library
into two sub-libraries. We used reversed-phase HPLC and
LC–MS to analyze and identify the best binders from the
DCLs and we employed aniline as a nucleophilic catalyst to
ensure that the equilibrium is established faster than in the
absence of a catalyst.
The first library, DCL-1, consisted of the four hydrazides
5, 6, 10, and 12 (100 mm each), and bis-aldehyde 3 (50 mm) in
presence of 10 mm aniline and 2% DMSO in 0.1m sodium
acetate buffer at pH 4.6, thus resulting in the formation of 15
potential homo- and hetero-bis-acylhydrazones (excluding E/
Z isomers) and five mono-acylhydrazones in equilibrium with
the initial building blocks. We were able to detect all of the
homo- and hetero-bis-acylhydrazones by LC–MS analysis.
Upon the addition of endothiapepsin, we observed amplifi-
cation of the bis-acylhydrazones 13 and 14 by more than three
times compared to the blank reaction (Figure 3 and Figure S1
in the Supporting Information). We set up the second library,
DCL-2, using the five hydrazides 4, 7, 8, 9, and 11 (100 mm
each), and bis-aldehyde 3 (50 mm) under the same conditions,
giving rise to the formation of 28 potential homo- and hetero-
We envisaged the linking of 1 and 2 to afford an inhibitor
that should occupy the S1, S1’, S2, and S2’ pockets of
endothiapepsin and benefit from numerous protein–ligand
interactions, while maintaining/improving the LE. With the
[
30]
help of the molecular-modeling software Moloc, we linked
the mesityl moiety of 1 to the naphthyl moiety of 2 through an
acylhydrazone linker, which resides at the junction of the S2
and S2’ pockets and appeared to be a suitable linker.
Acylhydrazone-based DCC is attractive for medicinal
chemistry-based projects because the resulting products
feature both H-bond donors and H-bond acceptors and are
stable enough as drug candidates under acidic and physio-
logical conditions. In our previous studies, we demonstrated
that acylhydrazone-based DCC is compatible with endothia-
[
8,18]
pepsin.
Inspection of known co-crystal structures of
[
31]
[32]
endothiapepsin and hotspot analysis of the active site
suggested that both aromatic and aliphatic moieties can be
hosted in the S2’ pocket, since they benefit from hydrophobic
contacts with residues G37, L133, and F194. Based on our
molecular-modeling studies and evaluation of synthetic
accessibility, we designed and optimized a series of bis-
acylhydrazone-based inhibitors of endothiapepsin. A super-
imposition of a modeled potential bis-acylhydrazone-based
inhibitor and the parent fragments is shown in Figure 2. All of
the bis-acylhydrazones form H-bonding interactions with the
catalytic dyad and most of them occupy the S1, S2, S1’, and S2’
pockets, and maintain the binding mode of fragments 1 and 2.
2
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Scheme 1. Structures and retrosynthetic analysis of the designed bis-acylhydrazone inhibitors and their corresponding aldehyde (3) and hydrazide
4–12) precursors.
(
bis-acylhydrazones (excluding E/Z isomers) and seven mono-
acylhydrazones in equilibrium with the initial building blocks.
Upon addition of the protein, bis-acylhydrazones 15 and 16
were amplified by a factor of more than two compared to the
blank reaction (Figure 3 and Figure S2 in the Supporting
Information). We also constructed a large library, DCL-3,
using all nine hydrazides (4–12) and bis-aldehyde 3 and
observed amplification of the previously observed bis-acylhy-
drazones 13, 14, and 16 along with bis-acylhydrazones 17 and
18 (Figure 3 and S3 in the Supporting Information). We
identified a total of two homo- (13 and 16) and four hetero-
(14, 15, 17 and 18) bis-acylhydrazones from the three libraries
DCL-1–3 (Figure 3).
To determine the biochemical activity of the amplified bis-
acylhydrazones, we synthesized the two homo-bis-acylhydra-
zones 13 and 16 from their corresponding hydrazides 5 and 8
and the bis-aldehyde 3 (see Schemes S2 and S3 in the
Supporting Information). We determined their inhibitory
potency by applying a fluorescence-based assay adapted from
[
34]
an assay for HIV protease. Biochemical evaluation con-
firmed the results of our DCC experiments, which were
analyzed by LC–MS. Bis-acylhydrazones 13 and 16 indeed
inhibit the enzyme with IC50 values of 0.054 mm and 2.1 mm,
respectively (see Figure 4, and Figures S4 and S5 in the
Supporting Information). The potency of the best inhibitor
was increased 240-fold compared to the parent hits. The
experimental Gibbs free energies of binding (DG) and LEs,
derived from the experimental IC50 values using the Cheng–
Figure 3. Chemical structures of the bis-acylhydrazones identified from
three DCLs using LC–MS analysis.
Figure 4. IC50 inhibition curve of 13 (IC50 =54.5Æ0.5 nm) measured in
duplicate; the errors are given as the standard deviation (SD).
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[
35]
À1
Prusoff equation,
are DG(13) = À49 kJmol , DG(16) =
dated in the S1 pocket and engaged in offset p–p stacking and
CH–p interactions with F116 and L125, respectively. The
phenyl group of 13 binds in the S2 pocket and is involved in
hydrophobic interactions with I300 and I304. Like the mesityl
group of 1, the phenyl group of 13 also engages in an amide–p
interaction with the peptide bond connecting residues G80
and D81. The phenyl moiety in 13 is connected with two imine
functionalities, thus making the aromatic ring electron-
deficient, which presumably strengthens the amide–p inter-
À1
À34 kJmol , and LE(13) = 0.29, LE(16) = 0.25, which rep-
resents an improvement in DG values while preserving the
LEs compared to the parent fragments (Table 1).
Table 1: The IC50 values, ligand efficiencies (LE), and calculated and
experimental Gibbs free energies of binding (DG) for the parent
fragments and bis-acylhydrazone inhibitors.
[
a]
[a]
Inhibitors
IC50 [mm]
Ki [mm]
DG
kJmol
LE
[36]
À1
action compared to the electron-rich mesityl group in 1.
[
]
In this study, we have demonstrated for the first time that
the synergistic combination of fragment linking and DCC is
a powerful and efficient strategy for accelerating hit identifi-
cation and optimization to afford inhibitors of the aspartic
protease endothiapepsin. We exploited LC–MS analysis to
identify the best binders directly from the DCLs. The best
inhibitor exhibits an IC50 value of 54 nm, representing a 240-
fold improvement in potency. Subsequent soaking experi-
ments validated our in silico fragment linking. Our strategic
combination of computational and analytical methods holds
great promise for accelerating drug development for this
challenging class of proteases, and it could afford useful new
lead compounds. This approach could be also extended to
a large number of other protein targets.
1
2
12.8Æ0.4
14.5Æ0.5
0.054Æ0.0005
2.1Æ0.1
6Æ0.2
À30
À30
À49
À34
0.27
0.29
0.29
0.25
7Æ0.2
1
1
3
6
0.0254Æ0.0002
0.98Æ0.05
[a] The Gibbs free energies of binding (DG) and the ligand efficiencies
(
LEs) were derived from the experimentally determined IC50 values.
To validate the predicted binding mode of the linked
fragments, we soaked crystals of endothiapepsin with the
most potent inhibitor (13) and determined its crystal structure
(
PDB ID: 5HCT) in complex with endothiapepsin at 1.36 ꢁ
resolution. 13 binds to the S1, S1’, and S2 pockets and
addresses the catalytic dyad through its a-C amino group
(
Figure 5a). A part of this bis-acylhydrazone is not visible in
the electron-density map, thus implying disorder of this
substituent across multiple conformational states, which is in Acknowledgements
line with our modeling studies. In two plausible poses, the
unresolved portion of bis-acylhydrazone 13 would be oriented
towards the S2’ and S6 pockets of the enzyme or remain
solvent-exposed (Figure 5b).
The detailed binding mode of bis-acylhydrazone 13 is
shown in Figure 5a. The visible portion of 13 preserves the
binding mode of the initial hit 1 and forms four charged
H bonds with the catalytic dyad through its a-C amino group,
as well as an H bond with the carboxylate group of D81
through the NH of the indolyl moiety, which is accommo-
Funding was granted by the Netherlands Organisation for
Scientific Research (NWO-CW, VIDI grant to A.K.H.H.) and
by the Dutch Ministry of Education, Culture, Science
(Gravitation program 024.001.035).
Keywords: dynamic combinatorial chemistry ·
fragment-based drug design · inhibitors · proteases ·
X-ray diffraction
Figure 5. a) X-ray crystal structure of endothiapepsin co-crystallized with bis-acylhydrazone 13 (PDB ID: 5HCT). b) Superimposition of the crystal
[
33]
structure (violet) and modeled structures (yellow and cyan) of 13.
4
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Published online: && &&, &&&&
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5
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Angewandte
Communications
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Communications
Drug Design
Better together: The synergistic combi-
nation of fragment linking and dynamic
combinatorial chemistry represents
a powerful and efficient strategy for
accelerating hit identification and opti-
mization, with the potential to afford new
lead compounds. The most potent
inhibitor of the aspartic protease endo-
thiapepsin identified in this way exhibits
an IC50 value of 54 nm, which represents
a 240-fold improvement in potency com-
pared to the parent hits.
M. Mondal, N. Radeva, H. Fanlo-Virgꢁs,
S. Otto, G. Klebe,
A. K. H. Hirsch*
&&&& — &&&&
Fragment Linking and Optimization of
Inhibitors of the Aspartic Protease
Endothiapepsin: Fragment-Based Drug
Design Facilitated by Dynamic
Combinatorial Chemistry
6
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