Sensitized detection of inhibitory fragments and iterative development of non-peptidic protease inhibitors by dynamic ligation screening

Article abstract of DOI:10.1002/anie.200704594

(Chemical Presented) A potential anti-SARS drug has been developed by dynamic ligation screening (DLS), by which nucleophilic fragments are directed to the protein's active site by reversible reaction with an aldehyde inhibitor. Their inhibitory effect is detected by competition with a fluorogenic enzyme substrate. With this concept, low-affinity fragments binding specifically to the active site are quickly identified in a functional enzyme assay.

Full text of DOI:10.1002/anie.200704594

Angewandte
Chemie
DOI: 10.1002/anie.200704594
Medicinal Chemistry
Sensitized Detection of Inhibitory Fragments and Iterative
Development of Non-Peptidic Protease Inhibitors by Dynamic
Ligation Screening**
Marco Florian Schmidt, Albert Isidro-Llobet, Michael Lisurek, Adeeb El-Dahshan, Jinzhi Tan,
Rolf Hilgenfeld, and Jörg Rademann*
Dedicated to Professor Günther Jung on the occasion of his 70th birthday
The conventional approach to identify biologically active,
druglike small molecules is based on high-throughput screen-
ing (HTS) of chemical libraries. However, the composition of
large chemical libraries and their screening are time-consum-
ing and expensive endeavors;the success relies heavily on the
quality of the available libraries, and even the largest library
can span only a minute section of the virtual chemical space.
Therefore, over the past decade several strategies have been
proposed to facilitate the development process by using the
protein target as a template for ligand assembly.[1–3] The
binding of low-molecular-weight fragments has been detected
“directly” by NMR spectroscopy[2a,b] or X-ray crystallogra-
phy.[2c,d] These biophysical methods have been demonstrated
to provide low-affinity ligands as rational starting points for
the iterative development of potent protein binders. Alter-
natively, protein-binding molecules have been identified from
mixtures of compounds formed in dynamic equilibria. In the
presence of a protein the equilibrium was shifted, and the best
binding products were concentrated in the mixture and could
be detected by chromatography, mass spectrometry, or NMR
spectroscopy.[3a,b] The reported fragment-based methods have
in common that they detect binding, not biological activity.
Moreover, all these methods require large amounts of protein
and test compounds and suffer from the difficult, time-
consuming, and expensive detection of active compounds.
We envisioned that the detection of bioactive ligands
should be sensitized considerably if reversibly formed ligation
products compete in dynamic equilibrium with a fluorogenic
reporter substrate for an enzyme (Figure 1). This approach
would combine dynamic, target-assisted formation of inhib-
itory species and detection by a fluorescence-based screening
methodology;thus, we designated it dynamic ligation screen-
ing (DLS). In DLS, the application of chemically reactive
inhibitors as directing probes should enable the testing of
inhibitory fragments for a defined binding site on the protein
surface. Using an enzymatic reaction for fragment detection
amplifies the signals and thus reduces the required amount of
protein drastically. Finally, enzymatic detection with a fluo-
rescent reporter molecule should enable high-throughput
screening (HTS) in microtiter plates (MTPs);thus, for the
first time conventional HTS methodology could be employed
in fragment-based dynamic ligand development.
[*] M. F. Schmidt, A. Isidro-Llobet, Dr. M. Lisurek, A. El-Dahshan,
Prof. Dr. J. Rademann
Leibniz Institute forMolecularPharmacology (FMP)
Robert-Rössle-Strasse 10, 13125 Berlin (Germany)
Fax: (+ 49)309-406-2981
E-mail: rademann@fmp-berlin.de
Homepage: http://www.fmp-berlin.de/rademann.html
M. F. Schmidt, Prof. Dr. J. Rademann
Institute forChemistry and Biochemistry
Free University Berlin
Takustrasse 3, 14195 Berlin (Germany)
A. Isidro-Llobet
Institute forResearch in Biomedicine
Barcelona Science Park
University of Barcelona
Josep Samitier1–5, 08028 Barcelona (Spain)
Dr. J. Tan, Prof. Dr. R. Hilgenfeld
Institute of Biochemistry
Center for Structural and Cell Biology in Medicine
University of Lübeck
The SARS coronavirus main protease (SARS-CoV Mpro
;
SARS = severe acute respiratory syndrome) was selected as
the protein target to demonstrate the DLS approach. SARS-
CoV Mpro is a cysteine protease that is essential for replication
of the virus inside the infected host cell. Thus, it has been
proposed as a drug target for SARS and—owing to the
reported high homology among coronaviral main proteases—
also for other coronaviral infections.[5] Several irreversible
(covalent) peptide-based inhibitors of SARS-CoV have been
prepared and cocrystallized with the enzyme;however, only a
few reversible,[6] non-peptidic[7] inhibitors have been reported
to date.
Ratzeburger Allee 160, 23538 Lübeck (Germany)
[**] We wish to thank Samuel Beligny, Angelika Ehrlich, Franziska
Hinterleitner, Dagmar Krause, Jörn Saupe, Bernhard Schmikale, and
Walter Verheyen for technical support. We also acknowledge
Jeroen R. Mesters and Koen H. Verschueren for discussions. Work
at the FMP was supported by the DFG (Ra 895/2-5), by the
government of Catalunya (stipend to A.I.L.), and by Boehringer
Ingelheim Pharma. Work at Lübeck University was supported by the
DFG (Hi 611/4-1), the Schleswig-Holstein Innovation Fund, the
Sino–German Center for the Promotion of Research, Bejing (GZ
233-202/6), and the European Commission through its SEPSDA
project (SP22-CT-2004-003831). J.R. and R.H. thank the Fonds der
Chemischen Industrie for continuous support.
To establish DLS for site-directed identification of
inhibitory fragments, at first a fluorescence-based assay[4] for
SARS-CoV Mpro activity was developed by employing the
Supporting information for this article is available on the WWW
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Table 1: Observed initial velocities v0 of the substrate conversion in the
presence of the SARS main protease, substrate, peptide aldehyde 2, and
active nucleophilic fragments.[a]
Electrophile
Nucleophile
v0 [mmminÀ1
]
–
2
–
–
5.5Æ0.2
2.8Æ0.1
2
2
1.0Æ0.1
1.0Æ0.1
2
2
1.6Æ0.1
1.9Æ0.1
2
2
2
2.1Æ0.1
2.2Æ0.1
2.2Æ0.1
Figure 1. The concept of dynamic ligation screening (DLS). Substrate 1
competes with peptide aldehyde inhibitor 2 forthe SARS-CoV main
protease (blue). Active fragment 3 leads to an increased inhibition
through the binding of the imine ligation product to the active site.
substrate Ac-TSAVLQ-AMCA (1). Enzymatic cleavage of 1
[a] For reaction conditions, see the Experimental Section.
released
2-(7-amino-4-methyl-3-coumarinyl)acetamide,
which was excited at 380 nm for fluorescence detection at a
wavelength of 460 nm. Second, a peptide aldehyde inhibitor 2
was selected for the DLS and synthesized on the protected
oxazolidine resin.[6] This peptide aldehyde contains a C-
terminal glutamine residue and thus forms an equilibrium
between the aldehyde and its cyclic condensation product in
aqueous solution.[6] Treatment of aryl aldehydes with an
excess of various primary amines has been reported to form
imines as major components of the equilibrium in aqueous
solution, whereas aliphatic aldehydes such as 2 are not
converted into the imines as the major product.[8] Thus, it
remained to be tested whether the hypothetical ligation
products of peptide aldehyde 2 and nucleophiles are stabi-
lized on a protein surface and consequently can be detected
by substrate competition.
For this purpose a collection of 234 nucleophiles was
assembled comprising aromatic and aliphatic amines, thiols,
and hydrazines. Aldehyde 2 as the directing probe was
incubated with an eightfold excess of one nucleophilic
fragment per well and in the presence of enzyme on a 384-
well microtiter plate. After the addition of reporter substrate
1, rate differences in the turnover of the substrate were
quantified to identify active inhibitory fragments (Figure 1,
Table 1). None of the selected fragments alone showed
activity as SARS-CoV Mpro inhibitor in a control experiment
at a concentration of 400 mm;thus, their affinity is in the
millimolar range or lower. For seven nucleophiles, however, a
stronger inhibition than with the inhibitor 2 alone was
observed (Table 1).
In the next step, the specific binding of identified hit
compounds to the active site of the SARS-CoV main
protease, and not, for example, to an allosteric site, had to
be confirmed. 3-Amino-(N-3-aminophenyl)benzamide (3)
was the most active and was selected for exemplary verifica-
tion of the binding site by combining chemical synthesis and
modeling. The imine formed from 2 and 3 was expected to be
the active species. To test this hypothesis, at first the reduced
amination product (4, Scheme 1) was synthesized. Tested in
the HPLC assay described by Tan et al.,[11] 4 displayed a KI
value of 50.3 mm. Comparison of the inhibitory activity of 4
with that of reduced amide 5 and those of the peptides Ac-
DSFDQ-OH, DSFDQ-OH, and Ac-DSFDQ-NH2, which all
were completely inactive at 500 mm, supported the directing
effect of peptide aldehyde 2 and the binding of fragment 3 to
the S1’ site. The lower inhibition by 4 compared to peptide
aldehyde 2 can be attributed to the absence of the electro-
philic carbonyl group interacting favorably with the active-
site cysteine residue of SARS-CoV Mpro. Furthermore, the
complexes of peptide aldehyde 2 and of the imine formed
with fragment 3 with SARS-CoV main protease were
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Figure 2. Molecularmodel of the aldehyde Ac-DSFDQ-H (A; residues
P5–P1) and the imine ligation product of Ac-DSFDQ-H with 3 (B)
docked into the substrate-binding site of the SARS-CoV Mpro (C cyan,
N blue, O red). The active site Cys145 is shown as a yellow surface.[10]
To obtain an entirely non-peptidic inhibitor of SARS-
CoV Mpro targeting both the S1’ and S1 pockets, the dynamic
ligation screening was conducted iteratively in a “reverted”
mode (Table 2). Instead of peptide aldehyde 2, which binds to
the S side of the binding cleft, 2-ketoaldehyde 9, which
presumably binds to the S’ side, was employed as a directing
Table 2: From nucleophilic fragment 3, the electrophilic 2-ketoaldehyde
inhibitor 9 was designed and used for “reverted” dynamic ligation
screening (observed initial rates of substrate conversion (v0) in the
presence of active nucleophiles).[a]
Nucleophile
Electrophile
v0 [mmminÀ1
]
–
11
9
9
4.3Æ0.1
Scheme 1. Development of a non-peptidic SARS-CoV Mpro inhibitor
through dynamic ligation screening. Active fragment 3, which binds to
the S1’site of the protein, has been transformed into electrophilic
derivatives 6–9, which were employed iteratively in reverted DLS,
yielding the non-peptidic inhibitor 12.
2.0Æ0.05
9
2.5Æ0.05
3.7Æ0.1
modeled, which suggested a possible binding mode of frag-
ment 3 (Figure 2).
9
Additional evidence for the binding of fragment 3 in the
S1’ pocket was provided by the synthesis and testing of
aldehydes and 2-ketoaldehydes 6–9, which are all electro-
philic derivatives of 3 (Scheme 1). Compounds 6–9 were
designed with an electrophilic group to interact with the
active site cysteine of the protease. While 6 and 7 were
obtained by oxidation of the respective alcohols, the 2-
ketoaldehydes 8 and 9 were prepared by polymer-supported
C-acylation, decarboxylation, and oxidative cleavage from a
phosphane resin.[12] Indeed all designed mono- and bis-
electrophiles were active inhibitors of SARS-CoV Mpro
(Scheme 1). Inhibitors 7 and 9, which are expected to position
the active fragment in the same place relative to the cysteine
residue as in the initial ligation product, were more potent
inhibitors than compounds 6 and 8. Benzaldehyde (10), used
as a control, was completely inactive, again indicating that the
fragments detected by DLS bind specifically to the S1’ pocket
[a] For reaction conditions, see the Experimental Section.
probe. For this experiment, 110 amines selected by diversity
analysis were screened. Compound 9 was incubated with one
amine per well, the protease, and the fluorogenic substrate
Ac-TSAVLQ-AMCA (1). In this second screen, three frag-
ments were identified which were active in the presence of the
directing probe 9 (Table 2). The most active was 11, which was
selected for verification of the inhibitor binding by chemical
synthesis. Using the 2-ketoaldehydes 8 and 9 for the covalent
linking appeared to be advantageous, as the aldehyde could
undergo reductive amination while the 2-keto functionality
remained intact for interaction with cysteine 145. Amine 11
was prepared as reported,[13] employed for reductive amina-
tion of 2-ketoaldehyde 9 with trichlorosilane as reducing
agent,[14] and yielded successfully 2-aminoketone 12 as the
of SARS-CoV Mpro
.
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Communications
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covalent ligation product. Compound 12 inhibited SARS-
CoV Mpro in the HPLC assay[11] with a KI value of 2.9 mm.
Thus, we can conclude that dynamic ligation screening
(DLS) enables the sensitized and site-directed detection of
low-affinity fragments with inhibition constants in the milli-
molar range that are difficult or impossible to detect with
previous dynamic strategies and conventional fragment-based
methods. The method was operated in high-throughput
format, and only very small amounts of protein were used
by exploiting the amplification effect of the enzyme-catalyzed
detection. No additional equipment was required besides a
standard microtiter plate reader. Most importantly, DLS was
operated iteratively in an evolutionary process and succeeded
in the transformation of a moderately active peptidic inhibitor
to an entirely non-peptidic inhibitor with an inhibition
constant in the low micromolar range. Dynamic ligation
screening has been demonstrated for protease inhibitor
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[9] The deviation was determined by plotting the observed fluores-
cence rate of three independent measurements in the program
“Prism Graph Pad”.
[10] For the modeling, the crystal structure of the reaction product of
SARS-CoV Mpro with a chloromethylketone was used as a
template (molecule A of PDB coordinate set 1K4;referen-
ce [7a]). Subsites S2, S1, and S1’ are labeled. Note that the
aspartate side chain in position P2 of the inhibitor is oriented
towards the solvent, while the phenylalanine residue in P3
occupies the hydrophobic S2 pocket of the enzyme. A similar
binding mode has been seen in the 1K4 structure by X-ray
crystallography (reference [7a]). The models were energy
refined with the program Sybyl. The figure was prepared using
Pymol (DeLano Scientific LLC, San Carlos).
Experimental Section
The activity of SARS-CoV Mpro was determined by measuring the
release of AMCA from the fluorogenic substrate Ac-TSAVLQ-
AMCA (1). The excitation wavelength was set to 380 nm, and the
emission was recorded at 460 nm. Relative fluorescence units (RFUs;
lem 460 nm) were determined as 63.861 RFUmm(AMCA)À1. Reaction
mixtures for cleavage were incubated at 298 K and contained 1 mm
SARS-CoV Mpro, 100 mm b-morpholinoethanesulfonic acid (MES)
buffer pH 7.0, and different concentrations of the fluorogenic
substrate (0.25 mm–2.5 mm) in a total volume of 20 mL. All measure-
ments were carried out on a TECAN SAFIRE fluorescence plate
reader (Crailsheim, Germany).
Dynamic ligation screening for the S1’ site was performed for a
library of 234 nucleophilic fragments using 1 mm of SARS-CoV Mpro
,
200 mm 1, 400 mm of one nucleophilic fragment per well, and 50 mm of
the peptide aldehyde inhibitor Ac-DSFDQ-H (2) on a 384-well
microtiter plate. The initial rates were observed and compared with
the initial rate without any nucleophilic fragment. Dynamic ligation
screening for the S1 site was performed for a library of 110
nucleophilic fragments using 1 mm of SARS-CoV Mpro, 200 mm 1,
200 mm of a nucleophilic fragment, and 20 mm of the non-peptidic
inhibitor 9 in a total volume of 20 mL MES buffer (100 mm, pH 7.0) on
a 384-well microtiter plate. The initial rate of product formation was
observed and compared with the initial rate of the controls.
Received: October 4, 2007
Published online: March 17, 2008
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Rao, J. Bigalke, B. Heisen, J. R. Mesters, K. Chen, X. Shen, H.
Jiang, R. Hilgenfeld, J. Mol. Biol. 2005, 354, 25 – 40.
Keywords: combinatorial chemistry · dynamic chemistry ·
enzyme catalysis · high-throughput screening ·
medicinal chemistry
.
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