Fragment-Based de Novo Design Reveals a Small-Molecule Inhibitor of Helicobacter Pylori HtrA

Article abstract of DOI:10.1002/anie.201504035

Sustained identification of innovative chemical entities is key for the success of chemical biology and drug discovery. We report the fragment-based, computer-assisted de novo design of a small molecule inhibiting Helicobacter pylori HtrA protease. Molecu

Full text of DOI:10.1002/anie.201504035

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International Edition: DOI: 10.1002/anie.201504035
German Edition: DOI: 10.1002/ange.201504035
De Novo Design
Hot Paper
Fragment-Based De Novo Design Reveals a Small-Molecule Inhibitor
of Helicobacter Pylori HtrA**
Anna M. Perna, Tiago Rodrigues, Thomas P. Schmidt, Manja Bçhm, Katharina Stutz,
Daniel Reker, Bernhard Pfeiffer, Karl-Heinz Altmann, Steffen Backert, Silja Wessler, and
Gisbert Schneider*
Abstract: Sustained identification of innovative chemical
entities is key for the success of chemical biology and drug
discovery. We report the fragment-based, computer-assisted
de novo design of a small molecule inhibiting Helicobacter
pylori HtrA protease. Molecular binding of the designed
compound to HtrA was confirmed through biophysical
methods, supporting its functional activity in vitro. Hit
expansion led to the identification of the currently best-in-
class HtrA inhibitor. The results obtained reinforce the validity
of ligand-based de novo design and binding-kinetics-guided
optimization for the efficient discovery of pioneering lead
structures and prototyping drug-like chemical probes with
tailored bioactivity.
confirmed specific binding of the NCE to H. pylori HtrA.
Hit expansion on the designed small molecule led to the
currently best-in-class HtrA inhibitor with favorable phys-
icochemical properties, binding kinetics and functional activ-
ity. The results obtained validate automated ligand-based
de novo design as a prime concept for the pioneering
discovery of tailored chemical matter for new macromolec-
ular targets.
H. pylori is a Gram-negative bacterial pathogen coloniz-
ing about 50% of the worldꢀs population and constitutes a risk
factor for developing gastric/duodenal ulcers, as well as
gastric cancer.[4] With increasing resistance against our con-
ventional antibiotic armamentarium, there is an unmet need
for novel therapeutic agents capable of preventing disease
onset and progression. Additionally, host–pathogen interac-
tions need to be further understood by use of small-molecule
probes to enable the design of efficacious drugs.[5] HtrA is
a virulence factor secreted by H. pylori and other pathogens,
and plays an important role in cleaving E-cadherin and
disrupting epithelial cell-to-cell adhesion.[6] Though, the
adequate study of target biology and validation of HtrA as
a relevant clinical drug target has been precluded by the lack
of proper chemical tools. Current state of the art relies on
rhodanine derivative 1a (Figure 1),[7] a poorly soluble,
aggregating chemical entity with potential for conjugation
with macromolecules through Michael addition. In a first
attempt at finding analogues of 1a with amended structural
features, we performed computational similarity searching in
a collection of 3255508 commercially available compounds.
Using 1a as query, we identified 11 potential HtrA inhibitors
(Table S2 in the Supporting Information). We used SPR for
compound profiling and assessing their binding affinities to
HtrA. While the query 1a has a KD value of (27 Æ 4) mm,
compounds 1b [(12 Æ 2) mm] and 1c [(10 Æ 1) mm] showed the
highest affinities among the 11 selected analogues (Fig-
ure S22).
T
he success of future drug discovery heavily relies on the
sustainable identification of new chemical entities (NCEs).[1]
By complementing high-throughput compound screening and
conventional in silico methods, computer-assisted de novo
molecular design is emerging as a promising technology.[2] For
example, it has recently been applied to the rational design of
prototypical kinase inhibitors.[3] Herein we disclose the
de novo design of an innovative Helicobacter pylori high-
temperature requirement A (HtrA) serine protease inhibitor.
The computationally designed compound displays suitable
properties for further development and chemical biology
studies. It blocked functional HtrA-mediated cleavage of E-
cadherin and inhibited H. pylori transmigration in an in vitro
pathogenesis model. Surface plasmon resonance (SPR) and
saturation-transfer difference (STD) NMR data further
[*] Dr. A. M. Perna,[+] Dr. T. Rodrigues,[+] K. Stutz, D. Reker,
Dr. B. Pfeiffer, Prof. Dr. K.-H. Altmann, Prof. Dr. G. Schneider
Department of Chemistry and Applied Biosciences, ETH Zurich
Vladimir-Prelog-Weg 4, 8093 Zürich (Switzerland)
E-mail: gisbert.schneider@pharma.ethz.ch
T. P. Schmidt, Prof. Dr. S. Wessler
Department of Molecular Biology, Universität Salzburg (Austria)
Noteworthy, no key scaffold hops were achieved by
similarity searching, as the selected compounds, like query
1a, still contain potentially reactive moieties. This result
points to limitations of virtual screens as a feasible solution
for the multidimensional exploration of chemical space. Thus,
we hypothesized that computer-assisted de novo design might
be better suited for this purpose, and provide chemotypes that
mitigate the liabilities of 1a–c. While a similar approach had
previously been pursued for the generation of innovative
chemotypes with predictable target affinities,[8] scaffold hops
have rarely been attempted with the goal of fulfilling multiple
Dr. M. Bçhm, Prof. Dr. S. Backert
Department of Biology, Universität Erlangen-Nürnberg (Germany)
[+] These authors contributed equally to the work.
[**] This research was financially supported by a grant from the OPO
Foundation, Zurich. The Chemical Computing Group and Inte:Li-
gand provided software licenses. The work of S.B. is supported by
SFB-796 (project B10). The work of S.W. is supported by a grant P-
24074 from the Austrian Science Fund (FWF). Camilla Pimenta and
Sarah Haller are acknowledged for technical support.
Supporting information for this article (experimental details, addi-
tional and raw data) is available on the WWW under http://dx.doi.
org/10.1002/anie.201504035.
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governed by the ligandꢀs off-rate,
with key implications for drug effi-
cacy and selectivity. Compounds 1a
and 2 showed fast koff rates and short
residence times (Table 1), suggest-
ing only few strong and directed
interactions within the binding
pocket, that is, moderate enthalpic
contribution to binding. These
observations are in agreement with
allosteric inhibition kinetics, as
reported elsewhere.[13] Altogether,
the SPR data obtained for 2 advo-
cate an innovative mechanism of
action warranting further investiga-
tion.
We used 1H STD NMR spec-
troscopy as an orthogonal method
to gain insight into the molecular
Figure 1. Structures of 1a, its analogues 1b,c, and the de novo designed entity 2 with its
retrosynthetic analysis.
objectives, including the improvement of physicochemical
properties.
Using 1a as design template, we applied the reaction- and
fragment-based de novo design software DOGS (design of
genuine structures)[9] to computationally generate new HtrA
inhibitor candidates. The algorithm suggested 1707 unique
molecules, among which 826 had computed logS > À6 (MOE
v2011.10). We further processed the obtained candidates with
a 3D pharmacophore model generated with LigandScout
(v3.02) that captured features we considered as critical for
bioactivity (see the Supporting Information). From the
remaining 65 molecules, we selected compound 2 (Figure 1)
Figure 2. Affinity of compounds 1a and 2 to H. pylori HtrA from SPR
for synthesis based on its ranking position, the absence of
a potential Michael acceptor, profound structural dissimilar-
ity to the template (Tanimoto index = 0.26; ECFP4), and
unreported chemotype (according to SciFinder). We obtained
compound 2 through a two-step synthetic route. Suzuki
coupling of l-(4-boronophenyl)alanine with 5-bromo-2-
furoic acid afforded the required intermediate for subsequent
Paal–Knorr pyrrole synthesis (Figure 1). Importantly, 2 is
chemically stable unlike related fragments reported else-
where.[10]
Colloidal aggregation of small molecules often yields
false-positive target-engagement readouts in early drug-dis-
covery programs.[11] We did not detect colloidal aggregates of
2 in concentrations up to 100 mm. Conversely, dynamic light-
scattering measurements of 1a revealed critical colloidal
aggregation in concentrations between 25 and 50 mm. Hence,
the poor aqueous solubility of 1a in bioactive concentrations
may cause unspecific ligand–receptor interactions. We
employed SPR to further profile 2. Compared to 1a, the
de novo designed compound 2 exhibited similar affinity
(KD = (37 Æ 4) mm, Figure 2), but a higher ligand efficiency
(Table 1).
measurements.
Table 1: Summary of the binding-kinetics parameters measured for
H. pylori HtrA, and physicochemical properties of compounds 1a and 2.
Compound
KD [mm]
koff [sÀ1
]
t1/2 [s]
LE[a]
logP[b]
logS[b]
1a
2
27Æ4
37Æ4
1.4
1.8
0.5
0.4
0.22
0.24
4.8
3.5
À8.7
À4.3
[a] Ligand efficiency (=À1.4logKD/(number of non-hydrogen atoms)).
[b] Octanol–water partition coefficient P and aqueous solubility S
calculated with MOE 2011.10.
basis of binding of 2 to HtrA (see the Supporting Informa-
tion). 1H NMR reference spectra were recorded for com-
pound 2 and HtrA separately (Figure 3A,B), as well as the
off-resonance reference (Figure 3C) and STD spectrum (Fig-
ure 3D) for the mixture of compound 2 and HtrA. Saturation
transfer could be best identified for the phenylfuranyl moiety
(6.75–7.50 ppm) and, to some minor extent, the methylene
protons of compound 2. Together with an appropriate
negative control (b-methyl-d-glucoside, Figure 3E–H), this
result not only confirms direct binding, as previously mea-
sured by SPR, but also corroborates directed interactions of 2
with HtrA. Furthermore, the STD NMR spectra support the
fast off-rate of 2 and provide a rationale for advanced lead-
Understanding the kinetics of drug binding to macro-
molecular targets is of vital importance for lead optimization,
as both the on- (kon) and off-rates (koff) determine the
duration of a given direct pharmacological effect.[12] The
residence time (t1/2) within a binding pocket is chiefly
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Following the thorough assessment of 2 by biophysical
methods, we evaluated whether binding of the de novo
designed entity translated into functional inhibition of
H. pylori HtrA. Full-length 130 kDa E-cadherin is an HtrA
substrate and the soluble E-cadherin fragment of about
90 kDa has been associated with a broad range of cancer
types.[14] Efficient inhibition of HtrA-mediated E-cadherin
cleavage might therefore provide an original means for
preventing H. pylori infection. Western blot analysis
(Figure 4) revealed that compound 2 shows concentration-
Figure 4. Western blot analysis of recombinant E-cadherin cleavage by
H. pylori HtrA in the presence of compounds 1a and 2 at different
concentrations.
Figure 3. STD NMR experiments revealing the direct interaction
between compound 2 and H. pylori HtrA. A) 1H NMR spectrum of
HtrA ligand 2 (1 mm). B) 1H NMR spectrum of H. pylori HtrA (10 mm).
C) Off-resonance reference NMR spectrum of compound 2 (1 mm) in
the presence of HtrA (10 mm). D) STD NMR spectrum of compound 2
(1 mm) in the presence of HtrA (10 mm). E) Off-resonance reference
NMR spectrum of b-methyl-d-glucoside (1 mm) in the presence of
HtrA. F) STD NMR spectrum of b-methyl-d-glucoside (1 mm) as
a negative control; 10 mm HtrA. G) Off-resonance reference NMR
spectrum of compound 2 (1 mm) together with b-methyl-d-glucoside
(1 mm) in the presence of HtrA (10 mm). H) STD NMR spectrum of
compound 2 (1 mm) together with b-methyl-d-glucoside (1 mm) in the
presence of HtrA (10 mm). In B), C), E), G), and H) an impurity in the
protein batch was observed at 3.25–3.75 ppm. In addition to residual
water, the expected signals, and residual protons of deuterated solvent
([D6]DMSO), are also observable. The impurity is not visible in the
STD spectrum in (D) indicating that it does not interact with HtrA.
This spectrum clearly shows saturation transfer from HtrA to the
aromatic protons of compound 2 (6.75–7.50 ppm) and, to some minor
extent, a methylene proton (3.25 ppm). The spectra in (E) and (F)
demonstrate that no saturation transfer occurs between HtrA and b-
methyl-d-glucoside. The spectrum in (H) shows the observed satura-
tion transfer from HtrA to the aromatic protons of compound 2, in the
mixture between compound 2 and b-methyl-d-glucoside with HtrA,
indicating the selective saturation transfer to the binding ligand.
dependent behavior and starts inhibiting cleavage of recombi-
nant E-cadherin at a concentration of 10 mm (Figure 4,
lane 6). At 50 mm the proteolytic activity of HtrA is blocked
by 2 (Figure 4, lane 8), thereby supporting de novo design as
a chief tool for obtaining innovative chemotypes with potent
bioactivity profiles.
Having unveiled 2 as a lead structure with a tractable
profile as an HtrA inhibitor, we engaged in a hit-expansion
program. Specifically, we set out to: 1) acquire preliminary
structure–activity relationship (SAR) data; 2) probe key
pharmacophore features for HtrA inhibition; and 3) optimize
the ligand efficiency of 2 through the modulation of its
binding kinetics. Hence, we synthesized compounds 3–8
(Figure 5). In 3–5 we truncated different moieties of 2 to
assess the importance of the carboxylic acid groups and the
steric bulk, in both the eastern and western regions of 2, on
HtrA affinity and in vitro functional effect. Furthermore, with
6 and 7 we studied how the absence of the eastern carboxylic
acid but presence of different bulky substituents affects
bioactivity. With 8 we probed the effect of diverging
physicochemical properties of the heteroaromatic moiety on
target engagement. We conducted SPR and Western blot
experiments to profile all compounds. The collected data (see
the Supporting Information) suggest that the eastern carbox-
ylic acid group is important for HtrA affinity and prevention
of E-cadherin cleavage. Conversely, an ionizable heteroar-
structure optimization. Head-to-head comparison of the
de novo designed compound 2 to its template 1a through
STD NMR spectroscopy was unsuccessful because of poor
aqueous solubility of the latter (logS(1a) = À8.7 vs. logS(2) =
À4.3, Table 1).
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The designed compounds have the required physicochemical
profile.
Finally, we assessed H. pylori transmigration in vitro in the
presence of compounds 2 and 5 at 50 and 100 mm (Figure 6B).
A transwell migration assay[17] served as a model of microbial
pathogenesis by measuring pathogen transmigration across
a polarized epithelial layer of human gastric adenocarcinoma
MKN-28 cells (see the Supporting Information). In accord-
ance with the Western blot analyses, significant effects were
detected at 50 mm over 24 h. A strikingly dissimilar outcome
was obtained for both compounds against Campylobacter
jejuni HtrA, suggesting critical differences between the HtrA
homologues of the two pathogens that may explain selective
blockage of H. pylori transmigration across the model gastric
epithelium by 2 and 5 (Figure 6C).
The results of the present fragment-based de novo design
study leverage chemical-biology and early drug-discovery
programs applied to HtrA. Particularly, we revealed the best-
in-class HtrA inhibitor to date through multi-objective
prioritization and binding-kinetics-guided optimization. The
outcome of this study corroborates computer-assisted
de novo design as a prime technology for obtaining innovative
chemical probes with desired properties.
Figure 5. Analogues of 2 obtained in hit expansion.
omatic system, for example, 8, is apparently detrimental,
resulting in a sharp loss of activity. Of note, the SPR
sensorgrams obtained for compound 8 advocate a different
mode of binding compared to 2–7 as no steady state was
observed (Figure S23). Similar findings have been reported
for two-step calcium-dependent protein–protein interac-
tions.[15]
As determined by SPR, our new lead compound 5 has
a KD value of (13 Æ 2) mm, that is, a 3-fold affinity improve-
ment over 2, and appropriate ligand efficiency (LE = 0.30;
Figure 6A). This improvement was chiefly governed by
a slower off-rate and longer residence time (koff = 8 ”
10À3 sÀ1; t1/2 = 89 s). Interestingly, an additional binding event
occurs at concentrations higher than 40 mm, which may
deserve further studies. These results were corroborated by
inhibition of E-cadherin cleavage at concentrations as low as
5 mm (see the Supporting Information) and the absence of
colloidal aggregation at 194 mm over the course of 1 hour
(kinetic solubility > 60 mgmLÀ1),[16] suggesting specific target
binding and suitable solubility. For further development it
should be kept in mind that H. pylori HtrA is an extracellular
protein and efficient inhibitors will require solubility in the
gastric microenvironment without membrane permeation.
Keywords: biophysical methods · chemical biology ·
computer-assisted drug design · drug design · receptor–
ligand interactions
How to cite: Angew. Chem. Int. Ed. 2015, 54, 10244–10248
Angew. Chem. 2015, 127, 10382–10386
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cells over a period of 24 h. Compounds 2 and 5 were tested at concentrations of 50 and 100 mm. C) Inhibition of wild-type Campylobacter jejuni
transmigration over a period of 24 h. Compounds 2 and 5 were tested at concentrations of 50 and 100 mm (N=3 independent experiments).
Significance at 24 h was analyzed with one-way ANOVA (Dunnett test), in comparison to untreated control (solid boxes): (*) p<0.05; (**)
p<0.01; cfu=colony-forming units.
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Received: May 3, 2015
Published online: June 9, 2015
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