Screening for covalent inhibitors using DNA-display of small molecule libraries functionalized with cysteine reactive moieties

Article abstract of DOI:10.1039/c6md00242k

DNA-encoded chemical libraries are increasingly used to identify leads for drug discovery or chemical biology. Despite the resurging interest in covalent inhibitors, libraries are typically designed with synthon filtered out for reactive functionalities that can engage a target through covalent interactions. Herein, we report the synthesis of two libraries containing Michael acceptors to identify cysteine reactive ligands. We developed a simple procedure to discriminate between covalent and high affinity non-covalent inhibitors using DNA display of the library in a microarray format. The methodology was validated with known covalent and high affinity non-covalent kinase inhibitors. Screening of the library revealed novel covalent inhibitors for MEK2 and ERBB2.

Full text of DOI:10.1039/c6md00242k

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Screening for covalent inhibitors using DNA-display of small
molecule libraries functionalized with cysteine reactive moieties†
C. Zambaldo,a J.-P. Daguer,a J. Saarbach,a S. Barluenga,a N. Winssinger*a
Received 00th January 20xx,
Accepted 00th January 20xx
DNA-encode chemical libraries are increasingly used to identify leads for drug discovery or chemical biology. Despite the
resurging interest in covalent inhibitors, libraries are typically designed with synthon filtered out for reactive
functionalities that can engage a target through covalent interactions. Herein, we report the synthesis of two libraries
containing Michael acceptors to identify cysteine reactive ligands. We developed a simple procedure to discriminate
between covalent and high affinity non-covalent inhibitors using DNA display of the library in a microarray format. The
methodology was validated with known covalent and high affinity non-covalent kinase inhibitors. Screening of the library
revealed novel covalent inhibitors for MEK2 and ERBB2.
DOI: 10.1039/x0xx00000x
www.rsc.org/
libraries aim to combine synthons that yield a final library with
desirable drug-like properties.11 These designs generally filter
out potentially reactive functionalities.
Introduction
Despite the notable success of covalent inhibitors in our
pharmacopeia (β-lactam, aspirin, omeprazole, clopidogrel for
instance), there is
a historic reluctance to pursue the
Results and discussion
development of compounds containing mildly reactive
functionalities that can engage a target through covalent
interactions.1 This is in contrast to natural products where
evolutionary pressure for bioactivity has frequently yielded
covalent inhibitors.2 In addition to natural products targeting
particularly activated residues within nucleophilic enzymes
(proteases and hydrolases), there is a plethora of natural
products that engage discrete nucleophilic residues in the
vicinity of a ligand-binding site. For instance, helenalin, which
is part of an extract of Arnica Montana, reacts with p65 thus
inhibiting the NF-κB pathway3 and is used topically to treat
inflammation. Another inspiring example is that of the
resorcylic acids that react with a discrete cysteine in the
nucleotide binding site of a subset of kinases.4 The challenge of
achieving sustained efficacy with some targets has recently led
to a resurging interest in covalent inhibitors,5 and two covalent
kinase inhibitors received FDA approval in 2013 (Afatinib,
Iburtinib).6 This rising interest in covalent inhibitors warrants
methodologies to identify covalent inhibitors in a primary
screen. In that respect, enzymatic assays are ill suited since
they do not discriminate between a reversible and covalent
inhibitor. DNA encoding technologies for small molecule
libraries are increasingly used in the discovery of novel drug
leads or probes for chemical biology.7-10 The majority of the
Several technologies have been developed to synthesize DNA-
encoded libraries: DNA-templated synthesis, split and mix
synthesis using DNA-tag ligation (enzymatic or chemical), or
hybridization to route the DNA tags.12-32 In addition, peptide
nucleic acid (PNA) encoding has been pursued on the basis
that its encoding chemistry utilizes SPPS that it is compatible
with co-synthesis of peptides and small molecule libraries.33-45
Several studies have established that hybridization of PNA-
tagged molecules onto a DNA microarray can be used to
rapidly identify the best binders within a library (Fig. 1).36, 44-50
We reasoned that this method could allow us to discriminate
between covalent and non-covalent ligands using a denaturing
wash that would denature proteins and compromise non-
covalent ligand-protein interactions. However, aside from
peptide-based libraries targeting proteases,33, 37-38 no DNA or
PNA-encoded library specifically designed to engage diverse
protein targets in covalent interactions had been reported.
Herein we report two PNA-encoded libraries specifically
designed to contain functionalities that can engage
nucleophilic residues covalently.
The first library was designed to have two points of
diversity with the second set of synthon including diverse
Michael acceptors (library I, Fig. 2). This reactive moiety was
selected based on the fact that it is present in many natural
products and has been successfully used for designed covalent
inhibitors targeting cysteine residues.
The synthesis
loaded with
proceeded as shown in Scheme 1, using resin
1
the orthogonally protected (Fmoc/Mtt) bifunctional linker and
a 12 Å PEG linker to distance the small molecule from its PNA
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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Journal Name
tag. The resin was split in 100 pools, and loaded with 100 of these 52 pools (25) was engaged in a SNAr with diverse
diverse Fmoc-protected amino acids (see Fig. for quinazolines (2 synthons) that were further each diversified by
1
representative examples). The Mtt group was removed (HFIP) palladium-catalyzed Suzuki coupling with 10 and 15 diverse
and the structure of the diverse amino acids was encoded with boronic acids respectively thus yielding 25 unique
a 7mer PNA. The quality of the synthesis was verified by a combinations. The remaining 27 pools were acylated with
cleavage of an analytical sample of resin for MALDI analysis. diverse resorcylic acids (5 synthons). The synthons bearing an
The pools were combined and re-split into 100 pools. In order Alloc-protected aniline were deprotected and further
to avoid exposing the library containing the Michael acceptor diversified with Michael acceptors. Alternatively to the azide
to the encoding chemistry, the second codon was introduced reduction, 48 pools of resin 10 were engaged CuAAC couplings
first (7mer PNA terminated with a BocLys(Mtt)OH) and the with diverse alkynes (5 synthons) and the products were
Fmoc group on the amino acid was removed to introduce the further diversified through the aforementioned palladium
synthon bearing the Michael acceptor; 34 synthons were cross couplings or acylation chemistry. In addition, one pool
obtained from commercial vendors while the rest were contained
a benzylic chloride that was diversified by
generated by coupling 2-methylenepentenoic acid followed by nucleophilic displacement with a thiophenol. Each of the pool
a CuAAC51-52 with 2-azidoacetic acid and derivatization with 66 was subjected to an analytical cleavage and MALDI analysis to
diverse amines. The 100 pools were subjected to analytical confirm the execution of the chemistry. Pools that failed the
cleavage and MALDI analysis confirming that each pool had a quality control were re-prepared. The pools were mixed and
molecular weight distribution that was consistent with a full the library cleaved from the resin to obtain a second library of
synthetic conversion. The pools were mixed and the library 10 000 compounds with 27% of the library bearing a reactive
was then cleaved from the resin to obtain the 10 000- functionality targeting cysteines. In addition, we prepared
membered-library in solution.
four known kinase inhibitors conjugated to PNA (a covalent
A second library was designed to target more specifically EGFR/ERBB2 inhibitor: canertinib;58 two high affinity but
kinases based on the use of a core scaffold that binds the nocovalent pankinase inhibitor: dasatinib,59 a staurosporin-
hinge domain of kinases (quinazoline or resorcylic acid moiety based bisindole maleimide (BIM);60-61 and high affinity
of known kinase inhibitors53-56): Library II, Fig. 3. Co-crystal allosteric inhibitor of MEK: PD-032590162, see Fig. 4 for
structures of resorcylic acid lactones with kinases57 have structures) as controls to establish the screening protocol. For
shown that the 2-hydroxybenzoic moiety occupies
a
the controls, the PNA tag was functionalized with a Cy3
comparable space and is involved in a similar network of fluorophore to visualize its hybridization and ensure that the
interactions as the quinazoline motif in kinase inhibitors. The compound is present on the microarray upon spiking within a
library was envisioned to have the same starting point as library. The Cy3 fluorophore used is spectrally resolved from
library I, Novapeg Rink resin bearing a Dap linker and a PEG the fluorophore (Dylight 649) used in protein detection (Cy5
spacer between the encoding region and the diversity region. channel).
The diverse scaffolds were planned to be connected to the first
In order to establish the conditions to discriminate
amino acid synthon using three different chemistries: between high affinity ligands and covalent ligands, we first
nucleophilic aromatic substitution (SNAr) for the quinazoline, investigated a screen against a therapeutically relevant kinase
classic amide coupling for the resorcylic acid analogs or a (ERBB2) and library I (Fig. 2) based on the availability of a
CuAAC51-52 to obtain a triazole in lieu of the amide on the control that binds covalently to this kinase (canertinib) and a
resorcylic acid scaffold. The last point of diversity was planned high affinity pankinase ligand (BMI). The array was self-
to arise from latent functionalities on the aryl moiety: aryl assembled by hybridization of the PNA-encoded library spiked
halide or suitably protected amines to be further derivatized with both controls onto a DNA microarray (Agilent custom
by Suzuki-reaction or acylation with Michael acceptors array, design 048196) under standard conditions.36,
44
The
respectively. Based on the fact that SNAr reaction and Suzuki array was then treated with the His-tagged ERBB2 in buffer (50
reaction had never been used in PNA-encoded library mM Hepes pH 7.4, 5 mM MgCl2, 5 mM MnCl2, 0.05% Tween
synthesis, all the synthons used were validated on the resin 20) for 60 min, washed with the same buffer and treated with
with a PNA tag to verify the efficacy of the chemistry (full a Dylight 649 anti-HisTag antibody. A scan in the Cy3 channel
synthetic details can be found in the Supporting Information). confirmed that the controls were at the preprogrammed
Starting with
1 (scheme 2), the resin was split into 100 pools position based on the complementarity of their PNA tags with
and each one was reacted with a unique amino acid, the Fmoc the DNA sequence on the array. A scan in the Cy5 channel
group was then removed and the resulting amine converted to indicated that there were several binders, including the two
an azide using imidazole-1-sulfonyl azide hydrochloride (
7) to positive controls: the covalent inhibitor (canertinib: position:
obtain 8. Each pool was encoded with a 7mer PNA (9) and the 1-2, Fig. 4A) and high affinity but non-covalent ligand (BMI:
resins were combined and re-split into 100 new pools. Each of position 79-75, Fig. 4A). In parallel, the same screen was
these pools was taken through a unique set of transformations performed followed by a wash using more stringent conditions
to afford 100 combinations that were encoded with a 7mer with a denaturing buffer (PBS, 1% SDS for 15 min with gentle
PNA (see scheme 2 for structures of individual fragments). rotation), Fig. 4B. It is important to note that despite the
Thus, 52 out of the 100 pools were treated with tris(2- stringent conditions, the PNA-DNA interaction remains
carboxyethyl)phosphine (TCEP) to reduce the azide. A subset unaffected and this point was verified by comparing the Cy3
2 | J. Name., 2012, 00, 1-3
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intensities corresponding the controls. Comparison of the Again, the non-covalent ligands were not detected but the
intensity of the signal of the two controls in the Cy-5 channel positive control, canertinib was amongst the strongest hits
revealed a significant loss of intensity for the non-covalent (Fig. 7). Amongst the other compounds identified, two
control (BIM) using the stringent wash while the covalent fragments (88 and 98) emerged as the fittest (Fig. 7). These
control retained its intensity (six fold difference in the ratio of fragments had been used in the development of a covalent
fluorescence intensity across both controls). Washing further inhibitor of EGFR, a kinase with a cysteine at the same position
the microarray from the non-stringent wash (Fig. 4A) without as ERBB2.66 Three compounds were selected as representative
SDS failed to remove the non-covalent interactions. However, examples and re-synthesized with a fluorophore (Cy3-PEG) in
more stringent conditions (higher concentration of SDS or lieu of the PNA (14-98, 20-98, 32-98). As shown in Fig. 8,
higher temperature) in the stringent wash led to loss of incubation of the compounds with ERBB2 for 60 min followed
PNA/DNA hybridization. Taken together, these results indicate by SDS denaturing gel clearly showed that three of the
that we have successfully identified conditions that can compounds labeled the protein in a covalent fashion.
discriminate between covalent and non-covalent interaction in
Finally, the selectivity of compound 32-98 lacking a tag was
a primary screening on the array. The experiment was assessed in a displacement assay against a broad panel of
repeated with MEK2, spiking the library with the four controls kinases (Kinome scan, DiscoverX). As shown in Fig. 9, the
(dasatinib, BIM, two ligand known to interact with MEK2;63 compound showed a very good selectivity, with two kinases
PD-0325901, an allosteric ligand specific for MEK2; canertinib, bearing a cysteine at the same position (ERBB2 and JAK3). In
a covalent ligand that should not react with MEK2). As shown addition, this compound also targeted MEK4 and DAPK3.
in Fig. 4C, the screen under mild washing condition clearly
shows the interaction of dasatinib (position 7-50), BIM
Conclusions
(position 79-75) and PD-0325901 (position 99-99) with MEK2.
It should be noted that MEK2 has cysteine residues in the
vicinity of the nucleotide binding site,64 but their positions are
not suitable to be engaged in a reaction with canertinib.
Indeed, the screening result does not show any binding of
canertinib despite the presence of the Michael acceptor.
Other compounds in the library were identified as potential
strong binders. Under the stringent washing conditions with
SDS to exclude non-covalent interactions (Fig. 4D), the positive
controls that interact non-covalently are now below the
detection threshold. Interestingly, there remained high
intensity binders suggesting that the library contains
compounds that successfully engaged MEK2 in a covalent
interaction (See Fig. 5 for highlighted compounds and their
structure). Six compounds were resynthesized, replacing the
PNA tag with a fluorophore (PEG-Cy3, Fig. 5B) in order to
validate their covalent engagement of MEK2. As shown in Fig.
5C, incubation of the compounds with MEK2 for 1h followed
by SDS denaturing gel showed that the compounds resulted in
a covalent adduct albeit differences in the reaction levels, with
compounds 69-7 and 97-7 being most efficient.
In conclusion, we developed
a simple procedure to
discriminate between covalent and high affinity non-covalent
inhibitors using DNA display. Notably, a stringent washing
with SDS can be used to remove non-covalent binders, despite
their strong target binding (low nM) without compromising the
nucleic acid hybridization. Based on the resurging interest in
covalent inhibitors and the growing implementation of DNA
encoded libraries in the identification of leads, this
methodology fills an important gap in the screening of DNA-
encoded libraries. While the present screening was performed
on microarrays, which facilitates the turnaround in the analysis
of results, we anticipate that it can also be used with affinity
selection for immobilized targets using next generation
sequencing technologies to identify the best inhibitors from a
library. The synthetic methodologies developed for the library
synthesis add to the repertoire of robust reactions for
diversification of PNA-encoded libraries and, demonstrate that
cysteine reactive functionalities can be incorporated in
encoded libraries. We anticipate that the results presented
herein will encourage the discovery of novel covalent
inhibitors from DNA or PNA encoded libraries.
To further validate that the compounds reacted with the
cysteines in the nucleotide-binding site, competition assays
pre-incubating the protein with a known covalent inhibitor
targeting the nucleotide binding site (NW-466)65, and with the
allosteric inhibitor (PD-0325901) were performed. As shown in
Fig. 6A, both competitors inhibited the labeling of MEK2 with
69-7 and 97-7 indicating that the labeling results from an
interaction of compounds 69-7 and 97-7 with the nucleotide
binding site and that this interaction accelerates the formation
of the covalent adduct. Next, we measure the rate of labeling,
analyzing the extent of labeling following a denaturing quench
of the reaction at different time points (Fig. 5B), showing a
half-life of reaction in >10 min in both cases.
Acknowledgements
The Swiss National Science Foundation, the NCCR in Chemical
Biology and the university of Geneva supported this work. The
authors thank Jose Garcia and Ganesan Karthikeyan for their
technical assistance.
Notes and references
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Applying the same screening methodology with library II
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Fig. 1 Discovering covalent binders for irreversible inhibition.
Fig. 2 Design of a generic PNA-encoded library of Michael acceptors, library I (entire set of fragments can be found in the supporting information).
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Scheme 1 Synthesis of a generic 10K PNA-encoded library of covalent binders (library I).
Fig. 3 Design of a kinase targeting PNA-encoded chemical library (library II), (full description can be found in the
supporting information).
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Fig. 4 Microarray screening with generic 10K PNA-encoded library of covalent binders (Library I) against A) 100nM ERBB2-his tagged with a 15
min PBS wash; B) 100nM ERBB2-his tagged with a 15 min SDS wash; C) 150nM MEK2-his tagged with a 15 min PBS wash and D) 150nM
MEK2-his tagged with a 15 min SDS wash. Controls in ERBB2 screens are Canertinib (1-2) and BIM (79-75). Controls in MEK2 screens are
Dasatinib (7-50), BIM and PD-0325901 (99-99). Controls are Cy3 labeled and the proteins were scanned using a Cy5 labeled antibody.
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Fig. 5 A) Microarray screening with generic 10K PNA-encoded library of covalent binders (Library I) against 150nM MEK2-his tagged with a 15
min SDS wash (expended view of Fig 4 D with highlighted hits). Selected compounds to synthesize off-oligo for hit validation. B) Compounds
resynthesized off-oligo as Cy3 conjugates. C) Scan of a SDS gel of MEK2-his 500nM labeling with Cy3 conjugates at 2hM in buffer (50 mM
Hepes, pH 7.4, 150 mM NaCl, 3mM MgCl2, 3mM MnCl2, 0.01% Tween 20, 10 hg of HEK cell extract) for 1 h at RT followed by 5 min
denaturation at 95 0C/SDS.
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Fig. 6 A) SDS gel scan of the MEK2-his 500nM labeling when the enzyme was pre incubated with the corresponding competitor (no
competitor, a hypothemycin analog NW-466 or PD-0325901) for 30 min at RT followed by incubation with Cy3 conjugates at 2hM in buffer
(50 mM Hepes, pH 7.4, 150 mM NaCl, 3mM MgCl2, 3mM MnCl2, 0.01% Tween 20, 10 hg of HEK cell extract) for 1 h at RT followed by 5 min
denaturation at 95 0C/SDS/10 mM DTT. B) SDS gel scan of MEK2-his 500nM labeling with compounds 69-7 and 97-7 Cy3 conjugate at 2hM in
buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 3mM MgCl2, 3mM MnCl2, 0.01% Tween 20, 10 hg of HEK cell extract) at RT followed by 5 min
denaturation at 95 0C/SDS/10 mM DTT, at different time points to study the kinetics of the covalent binding. The kinetics of labelling was
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Fig. 7 Microarray screening with kinase targeted PNA-encoded chemical library (Library II) against 100nM ERBB2-his tagged with a 15 min
SDS wash. Controls in the screen is e Canertinib-Cy3 encoded with codons 1-2 spiked in the library. Selected compounds resynthesized off-
oligo as Cy3 conjugates.
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Fig. 8 SDS gel scan of ERBB2-his tagged at 500nM labeling with compounds 14-98, 20-
98, 32-98 Cy3 conjugate at 2uM in buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 3mM
MgCl2, 3mM MnCl2, 0.01% Tween 20, 10 hg of HEK cell extract) for 1 h at RT followed
by 5 min denaturation at 95 0C/SDS.
Fig.
9 KinomeScan™ kinase selectivity profile for 32-98. Unconjugated 32-98 was
profiled at a concentration of 1 µM, against a diverse panel of more than 400 kinases
by DiscoverX Corporation.
Table of contentn entry: Discriminating between non-covalent and covalent
inhibitors with SDS wash in microarray-based screen.
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