Fragment-Based Drug Design Facilitated by Protein-Templated Click Chemistry: Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin

Article abstract of DOI:10.1002/chem.201603001

There is an urgent need for the development of efficient methodologies that accelerate drug discovery. We demonstrate that the strategic combination of fragment linking/optimization and protein-templated click chemistry is an efficient and powerful method that accelerates the hit-identification process for the aspartic protease endothiapepsin. The best binder, which inhibits endothiapepsin with an IC₅₀value of 43 μm, represents the first example of triazole-based inhibitors of endothiapepsin. Our strategy could find application on a whole range of drug targets.

Full text of DOI:10.1002/chem.201603001

DOI: 10.1002/chem.201603001
Communication
&
Drug Design
Fragment-Based Drug Design Facilitated by Protein-Templated
Click Chemistry: Fragment Linking and -Optimization of Inhibitors
of the Aspartic Protease Endothiapepsin
Milon Mondal+,[a] M. Yagiz Unver+,[a] Asish Pal,[b] Matthijs Bakker,[a] Stephan P. Berrier,[a] and
Anna K. H. Hirsch*[a]
fragment growing, linking, merging, or optimization. On the
one hand, fragment growing has become the optimization
strategy of choice,[7–12] even though it is time consuming be-
cause it requires synthesis and validation of the binding mode
of each derivative in the fragment–optimization cycle. To over-
come this hurdle, we have previously developed strategies in
which we combined fragment growing with dynamic combina-
torial chemistry (DCC) to render the initial stage of the drug-
discovery process more effective.[13] Fragment linking, on the
other hand, is very attractive because of its potential for super-
additivity (an improvement of ligand efficiency (LE) and not
just maintenance of LE), but challenging as it requires the pre-
servation of the binding modes of the individual fragments in
adjacent pockets and identification of the best linker with an
ideal fit.[14,15] It is presumably due to these challenges that
there are only few reports of fragment linking,[4,16] demonstrat-
ing the efficiency of linking low-affinity fragments to higher-af-
finity binders.[17–24] We have recently reported a combination of
DCC and fragment linking/optimization, which reduces the
risks associated with fragment linking.[25]
Abstract: There is an urgent need for the development of
efficient methodologies that accelerate drug discovery. We
demonstrate that the strategic combination of fragment
linking/optimization and protein-templated click chemistry
is an efficient and powerful method that accelerates the
hit-identification process for the aspartic protease endo-
thiapepsin. The best binder, which inhibits endothiapepsin
with an IC50 value of 43 mm, represents the first example
of triazole-based inhibitors of endothiapepsin. Our strat-
egy could find application on a whole range of drug tar-
gets.
Despite recent developments in medicinal chemistry, there is
a continuous need for the development of more efficient,
rapid, and facile strategies to accelerate the drug-discovery
process. In recent decades, fragment-based drug design
(FBDD) has emerged as an effective and novel paradigm in
drug discovery for numerous biological targets.[1–3] FBDD has
higher hit rates and better coverage of the chemical space, en-
abling the use of smaller libraries than those used for high-
throughput screening.[2] Since the first report of FBDD, it start-
ed to be more widely used in the mid-1990s[4] and has since
expanded rapidly. Over the course of the past two decades,
various pharmaceutical and biotechnology companies have
used FBDD and developed more than 18 drugs that are cur-
rently in clinical trials.[5]
In addition to DCC, protein-templated click chemistry (PTCC)
has emerged as a powerful strategy to design/optimize a hit/
lead for biological targets and holds the potential to reduce
the risks associated with fragment-linking.[26,27] PTCC relies on
the bio-orthogonal 1,3-dipolar cycloaddition of azide and
alkyne building blocks facilitated by the protein target.[28] This
highly exothermic reaction produces 1,4- and 1,5-triazoles,
which are extremely stable under acidic/basic pH as well as in
harsh oxidative/reductive conditions. Furthermore, triazoles
can participate in H-bonding, p–p-stacking, and dipole–dipole
interactions with the target protein and are a bioisostere of
amide bonds. In PTCC, the individual azide and alkyne frag-
ments bind to adjacent pockets of the protein and if the func-
tional groups are oriented in a proper manner, the protein
“clicks” them together to afford its own triazole inhibitor
(Figure 1). We have therefore envisaged that the potentially
synergistic combination of fragment linking and PTCC would
represent an efficient hit/lead identification/optimization ap-
proach in medicinal chemistry. Here, we have combined frag-
ment linking and PTCC by designing flexibility into the linker
and letting the protein select the best combination of building
blocks to identify a new class of hits for endothiapepsin, be-
longing to the pepsin-like aspartic proteases.
Upon identification of a fragment,[6] it has to be optimized
to a hit/lead compound and eventually to a drug candidate by
[a] Dr. M. Mondal,+ M. Y. Unver,+ M. Bakker, S. P. Berrier, Prof. Dr. A. K. H. Hirsch
Stratingh Institute for Chemistry, University of Groningen
Nijenborgh 7, 9747 AG Groningen (The Netherlands)
E-mail: a.k.h.hirsch@rug.nl
[b] Dr. A. Pal
Institute of Nano Science and Technology, Sector 64
Mohali, Punjab 160062 (India)
[+] These authors contributed equally to this work.
Supporting information and ORCIDs from the authors for this article are
available on the WWW under http://dx.doi.org/10.1002/chem.201603001.
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of Creative Commons Attri-
bution NonCommercial-NoDerivs License, which permits use and distribu-
tion in any medium, provided the original work is properly cited, the use is
non-commercial and no modifications or adaptations are made.
Aspartic proteases are a family of enzymes that are widely
found in fungi, vertebrates, and plants, as well as in HIV retro-
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3PBZ and 3PLD, respectively, Figure 2), identified by Klebe and
co-workers.[36] Both 1 and 2 are engaged in strong H-bonding
interactions with the catalytic dyad consisting of amino acid
residues D35 and D219, using their hydrazide and amidine
groups, respectively (Figure 2). Except for the number of H-
bond acceptors (four) for 1, both fragments 1 and 2 obey
Astex’s “rule of three”,[37] with a molecular weight (Mw) of 207
and 201 Da, three H-bond donors, four and two H-bond ac-
ceptors, two freely rotatable bonds and total polar surface
areas (TPSAs) of 58.4 and 49.9 ꢁ2, respectively. At a concentra-
tion of 1 mm, fragments 1 and 2 display 89 and 84% inhibition
of endothiapepsin, respectively. Considering their promising
physicochemical properties, inhibitory potency, their small size
(15 and 12 heavy atoms, respectively) and the fact that they
bind to adjacent pockets of endothiapepsin, we chose them as
a starting point for fragment linking/optimization into an in-
hibitor of endothiapepsin.
Figure 1. Schematic representation of protein-templated click chemistry
leading to a triazole-based inhibitor starting from a library of azides and al-
kynes.
viruses. This class of enzymes plays a causative role in several
important diseases such as malaria, Alzheimer’s disease, hyper-
tension, and AIDS.[29] Owing to its high degree of similarity
with these drug targets, endothiapepsin has served as a model
enzyme for mechanistic studies[30–32] as well as for the identifi-
cation of inhibitors of renin[33] and b-secretase.[34] Endothiapep-
sin is a robust enzyme, is available in large quantities, crystal-
lizes easily, and remains active at room temperature for more
than three weeks, making this enzyme a convenient represen-
tative for aspartic proteases.[35] All aspartic proteases consist of
two structurally similar domains, which contribute an aspartic
acid residue to the catalytic dyad that is responsible for the
water-mediated cleavage of the substrate’s peptide bond.[31,32]
Although the linkage of two known inhibitors of acetylcho-
linesterase via a triazolyl linker using PTCC has been reported,
the inhibitors that are linked do not qualify as fragments.[27] To
the best of our knowledge, there is no report of fragment link-
ing using PTCC. Herein, we describe how we combined frag-
ment linking/optimization and PTCC for the efficient fragment-
to-hit optimization of inhibitors of the aspartic protease endo-
thiapepsin.
Fragments 1 and 2 occupy the S3 and S1 and the S2 and S1’
pockets, respectively, and address the catalytic dyad using an
H-bonding network (Figure 2). With the help of the molecular-
modeling software Moloc[39] and the FlexX docking module in
the LeadIT suite,[40] we linked these two fragments using a tri-
azolyl linker. The newly introduced triazolyl moiety resides at
the junction of the S1 and S1’ pockets, where hydrazide and
amidine groups of fragment 1 and 2, respectively, were posi-
tioned. The triazolyl linker appeared to be ideally suited to ad-
dress the catalytic dyad through a H-bonding network. Al-
though the protonation of 1,2,3-triazole at pH 4.6, optimal for
endothiapepsin, is unprecedented, given that its pKa value in
water is 1.2,[41] in the active site of endothiapepsin, the triazole
is expected to bind in close proximity to the two Asp residues
(D35 and D219), which will modulate the pKa value, facilitating
protonation. pKa perturbation is a general phenomenon and
has been observed, for instance, in several co-crystal structures
of endothiapepsin in complex with heterocyclic fragments.[42]
Hence, under acidic conditions, one of the N atoms of the tri-
azole is likely protonated and engaged in a H-bonding interac-
tion with residue D35. Careful analysis of known co-crystal
structures of endothiapepsin[35,36] as well as hotspot analysis[43]
of the active site of endothiapepsin suggested that
We used X-ray crystal structures of endothiapepsin in com-
plex with fragments 1 and 2 (Protein Data Bank (PDB) codes:
the S2 pocket can host aromatic moieties, which can
be involved in hydrophobic interactions with residues
F194, I217, I304, and I300. The S3 pocket could ac-
commodate a piperazine ring instead of the tertiary
amine, which can be involved in an additional H-
bonding interaction with residue D119. On the basis
of molecular modeling and docking studies, we de-
signed and optimized a series of triazole-based inhib-
itors. A superimposition of a designed potential tri-
azole inhibitor and the two fragments is shown in
Figure 2. All of the triazoles are engaged in H-bond-
ing interactions with D35 and occupy the S3, S1, S1’
and S2 pockets, the binding sites of fragments 1 and
2.
Retrosynthesis of all designed triazole derivatives
Figure 2. X-ray crystal structure of endothiapepsin in complex with fragments 1 and 2
(PDB code: 3PBZ and 3PLD, respectively) and a modeled potential triazole inhibitor in
the active site.[36] Color code: protein skeleton: C: gray, O: red, and N: blue; fragment
skeleton: C: purple, yellow and green, N: blue, O: red, Cl: green. Hydrogen bonds below
3.0 ꢁ are shown as black, dashed lines.[38]
leads to nine azides (3–11) and the alkyne 12,
(Scheme 1). We also included alkynes 13–15 in our li-
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Figure 3. Structure of the triazoles (17–20) identified using PTCC, inactive tri-
azole 21.
To investigate the biochemical activity of the binders identi-
fied by PTCC, we synthesized all four triazoles from their corre-
sponding azide and alkyne precursors using the CuI-catalyzed
1,3-cycloaddition (Schemes S3–S6 in the Supporting Informa-
tion). In addition, we synthesized an inactive triazole 21 to
demonstrate the efficiency of PTCC (Figure 3 and Scheme S7 in
the Supporting Information). We determined their inhibitory
activity using a fluorescence-based assay adapted from the
HIV-protease assay.[46]
The enzyme-activity assay confirmed the result of the PTCC
experiment. Three out of the four triazoles indeed inhibit en-
dothiapepsin with IC50 values in the range of 43–121 mm (Fig-
ures S10–S12 in the Supporting Information). We were unable
to determine the IC50 value of 20 because of its poor solubility
even at 250 mm using the maximum possible DMSO concentra-
tion for the assay. The inactive triazole 21, which was not ob-
served in the PTCC but synthesized as a control, did not show
any activity in the enzyme-activity assay. The most potent tri-
azole inhibitor 17 displays an IC50 value of 43 mm (Table 1). The
experimental Gibbs free energies of binding (DG) and ligand
efficiencies (LE), derived from the experimental IC50 values
using the Cheng–Prusoff equation,[47] correlate with the calcu-
lated values using the scoring function HYDE in the LeadIT
Scheme 1. a) Structures and retrosynthetic analysis of the designed triazole
inhibitors starting from fragments 1 and 2; b) structures of the azides 3–11
and the alkynes 12–15.
brary, which were available from Syncom. While we obtained
all azides from their corresponding bromides by treatment
with sodium azide in 40–80% yield (Scheme S1 in the Support-
ing Information),[44,45] we synthesized alkyne 12 using a Sonoga-
shira cross-coupling reaction, starting from the iodide 16
(Scheme S2 in the Supporting Information).
We set up a library, consisting of four alkynes 12–15 (100 mm
each) and nine azides 3–11 (100 mm each), in the presence of
a catalytic amount of protein (26 mm) to investigate whether
the protein would select a pair of fragments from a library to
template the formation of a triazole binder with high affinity
(Scheme S8 in the Supporting Information). The advantage of
PTCC is that it accelerates the screening time to two weeks by
avoiding the synthesis of individual triazoles and reduces the
amount of protein required in each individual analysis.
We used UPLC-TOF-SIM (selective ion monitoring) to analyze
the formation of triazoles in the reaction mixture. SIM mea-
surements are highly sensitive. We monitored for [M+H]+ of
all potential triazole products present in the library. After incu-
bation of the protein at room temperature for two weeks (en-
dothiapepsin is stable and active during this time period),[35]
the library was analyzed using UPLC-TOF-SIM. To differentiate
between the two regioisomers of triazoles (1,4- and 1,5-tri-
azole), we set up two libraries using the same azide and alkyne
building blocks, once in the presence of CuI-catalyst to selec-
tively afford the 1,4-triazoles and once under Huisgen cycload-
dition conditions to obtain both 1,4- and 1,5-triazoles. We also
compared the PTCC reaction with the blank reaction (without
protein) as well as the protein alone. We identified a total of
four 1,4-triazoles (17–20), which are formed only in the pres-
ence of protein (Figure 3 and Figures S2–S9 in the Supporting
Information). To establish that the active site of intact endo-
thiapepsin is required for PTCC, we set up two control experi-
ments. Repeating the reaction in the presence of saquinavir
(100 mm, a strong inhibitor, Ki =48 nm), or a catalytic amount
of bovine serum albumin (BSA; 26 mm) did not lead to the for-
mation of any triazoles.
Table 1. The IC50 values, ligand efficiency (LE), calculated and experimen-
tal Gibbs free energies of binding (DG) of triazole inhibitors.
[a]
[b]
[c]
Inhibitors
IC50
DGEXPT
LE[b]
DGHYDE
[mm]
[kJmolÀ1
]
[kJmolÀ1
]
17
18
19
20
7
9
12
14
43Æ0
À27
À25
À24
–
–
–
0.25
0.26
0.22
–
–
–
À25
À19
À25
À23
–
–
–
–
94Æ18
121Æ3
insoluble
no inhibition
no inhibition
142Æ52
–
–
–
–
no inhibition
[a] 26 experiments were performed and only initial six experiments were
considered to calculate the initial slope (n=6), 11 different concentrations
of inhibitor were used, starting at 1 mm; each experiment was carried
out in duplicate and the errors are given in standard deviations (SD),
[b] The Gibbs free energy of binding (DGEXPT) and the ligand efficiencies
(LEs) derived from the experimentally determined IC50 values, [c] Values
indicate the calculated Gibbs free energy of binding (DGHYDE; calculated
by the HYDE scoring function in the LeadIT suite).
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suite (DGHYDE(17)=À25 kJmolÀ1, Table 1).[48,49] This correlation
is also valid for the other triazole inhibitors (Table 1).
involved in several hydrophobic contacts with I300, I302, I304,
F194, and I217, maintaining the binding mode of fragment 2.
Triazole 18 displays an IC50 of 94 mm and (S)-18 addresses
the catalytic dyad using its triazolyl linker to form a direct H
bond with D35, as indicated by modeling and docking studies
(Figure 4b). The ÀNH2 group of the triazole is engaged in H-
To validate the predicted binding mode from fragment link-
ing, we tried to soak crystals of endothiapepsin with the most
potent triazole inhibitor 17. Due to limited solubility, we were
not able to obtain crystals of 17 with endothiapepsin. Based
on the inhibitory potencies, replacement of ÀCl in 19 with a À bonding interactions with D33 and G221. Both phenyl sub-
OH group in 17, leads to a decrease in IC50 value from 121 to
43 mm. This result indicates that the ÀOH group is involved in
more favorable interactions than ÀCl, which could be due to
the H-bonding interaction with I300 in the S2 pocket, as illus-
trated by modeling studies (Figure 4a, and Figure S1 in the
stituents of the triazole (S)-18 occupy the S3 and S2 pockets
and are involved in hydrophobic interactions with F116, I122,
and L125 in the S3 pocket, and I300, I302, I304, F194, and I217
in the S2 pocket, which preserve the binding mode of frag-
ments 1 and 2, respectively.
In conclusion, we have demonstrated for the first time that
the strategic combination of fragment linking/optimization
and PTCC is an efficient and powerful method that accelerates
the hit-identification process for the aspartic protease endo-
thiapepsin. We have exploited the sensitive UPLC-TOF-SIM
method to identify the triazole binders templated by the pro-
tein. The best binder inhibits endothiapepsin with an IC50
value of 43 mm. Due to the limited solubility of the triazoles
identified, we were unable to obtain crystals of any triazole in
complex with endothiapepsin. We have reported the first ex-
ample of triazole-based inhibitors of endothiapepsin. The ad-
vantage of our approach is that, a catalytic amount of protein
is sufficient to initiate and accelerate triazole formation from
a sufficiently large library. Our strategic combination of meth-
odologies proved to be very successful for the hit identification
for the aspartic protease endothiapepsin and could be applied
to a wide range of biological targets. It could be used in the
early stages of drug development and holds the potential to
greatly accelerate the drug-discovery process.
Acknowledgements
We would like to acknowledge T. D. Tiemersma-Wegman for
help with the UPLC-TOF-SIM measurements, Prof. G. Klebe and
N. Radeva for fruitful discussions and help with protein crystal-
lization experiments, and Syncom for providing alkynes 13–15.
Funding was granted by the Netherlands Organisation for Sci-
entific Research (NWO-CW, ECHO-STIP grant to A.K.H.H.) and
by the Dutch Ministry of Education, Culture, Science (gravita-
tion program 024.001.035).
Figure 4. Moloc-generated modeled structures of: a) 17, and b) (S)-18 in the
active site of endothiapepsin. Color code: inhibitor skeleton: C: green,
violet, N: blue, O: red; enzyme skeleton: C: gray. H bonds below 3.2 ꢁ are
shown as black, dashed lines.
Supporting Information). Moreover, the alkyne 12 displays an
IC50 value of 142 mm (Figure S13 in the Supporting Information)
and is present in both 17 and 19, two identified triazoles. Frag-
ment 12 is a privileged fragment for endothiapepsin and most
probably the binding mode of 12 is retained in both 17 and
19.
Keywords: click chemistry
· drug design · enzymes ·
inhibitors · liquid chromatography
According to modeling and docking, as shown in Figure 4a,
and Figure S1, respectively, both 17 and 19 address the cata-
lytic dyad using their triazolyl linker to form direct H-bonds
with D35. The NH group of both compounds is involved in
a H-bonding interaction with D119 in the S3 pocket. The piper-
azinyl group of both triazoles occupies the S3 and part of the
S1 pockets and is engaged in hydrophobic interactions with
F116, I122, and L125, maintaining the binding mode of frag-
ment 1. The ÀCl and ÀOH substituted phenyl groups of tria-
zoles 17 and 19 occupy the S2 and part of S1’ pockets and are
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Published online on && &&, 0000
Chem. Eur. J. 2016, 22, 1 – 6
www.chemeurj.org
5
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers! ÞÞ

Communication
COMMUNICATION
&
Drug Design
M. Mondal, M. Y. Unver, A. Pal, M. Bakker,
S. P. Berrier, A. K. H. Hirsch*
&& – &&
Fragment-Based Drug Design
Facilitated by Protein-Templated Click
Chemistry: Fragment Linking and
-Optimization of Inhibitors of the
Aspartic Protease Endothiapepsin
The strategic combination of fragment
linking/optimization and protein-tem-
plated click chemistry is an efficient and
powerful method that accelerates the
hit-identification process for the aspartic
protease endothiapepsin. The highly
sensitive UPLC-TOF-SIM method has
been used to identify the triazole bind-
ers templated by the protein. The best
binder inhibits endothiapepsin with an
IC50 value of 43 mm.
&
&
Chem. Eur. J. 2016, 22, 1 – 6
www.chemeurj.org
6
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!