Affinity Enhancement of Protein Ligands by Reversible Covalent Modification of Neighboring Lysine Residues

Article abstract of DOI:10.1002/anie.201811650

The discovery of protein ligands, capable of forming a reversible covalent bond with amino acid residues on a protein target of interest, may represent a general strategy for the discovery of potent small-molecule inhibitors. We analyzed the ability of di

Full text of DOI:10.1002/anie.201811650

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Accepted Article
Title: Affinity enhancement of protein ligands by reversible covalent
modification of neighboring lysine residues
Authors: Alberto Dal Corso, Marco Catalano, Anja Schmid, Jörg
Scheuermann, and Dario Neri
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10.1002/anie.201811650
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Affinity enhancement of protein ligands by reversible covalent
modification of neighboring lysine residues
Alberto Dal Corso,# Marco Catalano,# Anja Schmid, Jörg Scheuermann and Dario Neri*
families, such as the full complement of PLP-dependent
Abstract: The discovery of protein ligands, capable of forming a
reversible covalent bond with amino acid residues on protein target
of interest, may represent a general strategy for the discovery of
potent small molecule inhibitors. We analyzed the ability of different
enzymes in Staphylococcus aureus.8
The use of functional groups, capable of forming a reversible
covalent bond with lysine residues, could be useful for
pharmaceutical applications, provided that the reactivity of those
chemical moieties was not too high, leading to a blockade by
human serum albumin in blood. In this Communication, we have
systematically analyzed the equilibrium constants for the
reaction of various benzaldehydes with primary amines. Using
four different protein targets, which display a different density of
lysine residues, we have investigated how the incorporation of
suitable aromatic aldehydes into the ligand structure can lead to
an affinity enhancement.
1
aromatic aldehydes to form imines by reaction with lysine using H-
NMR techniques. 2-Hydroxybenzaldehyde derivatives were found to
efficiently form imines in the millimolar concentration range. These
benzaldehyde derivatives could increase the binding affinity of
protein ligands towards the cognate protein target. Affinity
maturation was achieved not only by displaying ligand and aldehyde
moieties on two complementary locked nucleic acid strands, but also
by incorporating the binding fragments in a single small molecule
ligand. The affinity gain was only observed when lysine residues
were accessible in the immediate surroundings of the ligand binding
site and could be abrogated by quenching with a molar excess of
hydroxylamine.
Figure 1A depicts how Schiff base formation for benzaldehyde
derivatives may be in competition with the hydration of the
aldehyde moiety and how these equilibria can be perturbed by
neighboring functional groups. The two reactions can be
monitored and quantified using 1H-NMR methods [Figure 1B].
We observed that the least reactive benzaldehyde derivatives
[e.g., benzaldehyde (1)] exhibited no detectable Schiff base
formation at pH 7.4 in the presence of 20 mM Nα-Acetyl-lysine.
By contrast, efficient imine formation could be detected for 2-
hydroxybenzaldehydes [e.g., (13)], with an apparent dissociation
Ligands capable of covalent binding to protein targets of interest
are important for pharmaceutical applications and as chemical
biology tools.1,2 Aspirin, penicillin, omeprazole and clopidogrel
are examples of successful covalent drugs. Moreover, the
selective modification of cysteine residues close to the active
site of kinases with suitable Michael acceptors has led to the
development of covalent inhibitors for cancer therapy, such as
ibrutinib (BTK inhibitor, Imbruvica™; AbbVie), afatinib (EGFR
inhibitor, Gilotrif™; Boehringer) and osimertinib (EGFR inhibitor,
constant KD
= 23 mM [Figure 1C]. The ortho-formyl-
Tagrisso™;
AstraZeneca).
Recently,
researchers
at
phenylboronic acid (14) moiety, which had been used for the
generation of reversible covalent Mcl-1 inhibitors3, exhibited a KD
value of 17 mM, while phthaldialdehyde (16) strongly reacted to
form an irreversible 1-isoindolinone adduct.9
AstraZeneca have reported the generation of highly potent
inhibitors of induced myeloid leukemia cell differentiation protein
(Mcl-1), featuring a formyl- or an acetyl-phenylboronic acid
moiety in ortho position.3 This additional group is capable of
forming a reversible covalent bond with the ε-amino group of a
We have previously shown that the display of chemical moieties
on complementary DNA strands may facilitate the discovery and
characterization of synergistic binding fragments to protein
targets.10 For example, we and others have shown that the
potency and selectivity of Michael acceptors towards cysteine-
containing kinases may be increased by the incorporation of
binding fragments on complementary nucleic acids strands, with
a “dock and lock” mechanism.11-12
specific lysine residue.4,5 Imine formation is
reaction of amines and several enzymes take advantage of
a well-known
Schiff base formation with
a pyridoxal phosphate (PLP)
cofactor.6 The latter has been investigated as useful tool for
different applications in chemical biology. For instance, Francis
and coworkers had previously investigated the use of PLP and
ortho-hydroxy-pyridinecarboxyaldehydes
for
site-specific
modification of N-terminal amino acids.7 Moreover, PLP
derivatives have been recently designed to be integrated into the
cell metabolic processing, enabling the discovery of new protein
In this work, we initially assessed the contribution of
benzaldehyde derivatives to protein binding for various ligands,
using chemical derivatives of short (12-bp) locked nucleic acids
(LNAs) [Figure 2A].13 We tested our methodology on three
protein targets: human serum albumin (HSA), bovine carbonic
anhydrase II (CAII) and human interleukin-2 (IL2). For these
targets, we and others had previously described small molecule
ligands.14-18 The binding sites of these ligands on the cognate
proteins differed in terms of local density of lysine residues
[Figure 2B].
Department of Chemistry and Applied Biosciences
Swiss Federal Institute of Technology (ETH Zürich)
Vladimir-Prelog-Weg 4, CH-8093 Zurich (Switzerland)
E-mail: neri@pharma.ethz.ch
*) Corresponding author
#) Authors contributed equally to this work
Supporting information for this article is given via a link at the end of the
document.
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10.1002/anie.201811650
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Figure 1. Reversible covalent modification of primary amines with benzaldehyde derivatives. (A) Aldehyde hydration and Schiff base (imine) formation as
alternative reaction routes for benzaldehydes and other examples of imine formation assisted by functional groups in the ortho position; (B) 1H-NMR spectra of
the reaction products of benzaldehydes 9 and 13 (2 mM) with 20 mM of Nα-Acetyl-lysine (Ac-Lys-OH), performed at room temperature in phosphate buffer, pH =
7.4, dissolved in D2O]. While for 9 the aldehyde hydration reaction is predominant, compound 13 yields a stable imine product. (C) The percentage of hydration
product (red) and/or imine formation (blue) was quantified using 1H-NMR and used to measure KD constants. The chemical structures of the compounds and the
corresponding numbers are indicated. *Data referred to the integration of broad NMR signals; ** Data referred to the 100% aldehyde conversion, according to the
mechanism depicted in Figure 1A.
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A similar affinity gain (4-fold) was observed for the IL2 ligand,
but in this case also the benzaldehyde derivative 1-LNA
exhibited an improved dissociation constant compared to 0-LNA.
As expected, the binding potentiation of HSA and IL2 ligands
could be quenched by the addition of a molar excess of
hydroxylamine [Figure 3 and Supplementary Figure S1].
By contrast, no affinity gain could be detected for the CAII
ligands, as CAII lacks primary amines in close proximity to the
active site [Figures 2 and 3].
Figure 2. (A) Schematic representation of
a “dock-and-lock” procedure,
featuring bifunctional structures displayed at the extremity of a fluorescently-
labeled nucleic acid. In a first step, a non-covalent ligand interacts with the
cognate site on the target protein of interest. Subsequently, a benzaldehyde
moiety may form
a Schiff base with a neighboring lysine residue, thus
stabilizing the complex. (B) Chemical structures of ligands to human serum
albumin (HSA), carbonic anhydrase II (CAII) and interleukin-2 (IL2), whose
binding characteristics had previously been described.16-18 Space-fill
representations of the three-dimensional structures of the target proteins, with
lysine sidechains depicted in blue, reveals a high density of primary amines
around the active sites of HSA and IL2, but not for CAII. Ligands of HSA and
IL2 are depicted in green in the crystal structures, while only the zinc atom in
the active site is depicted in green for CAII (Brookhaven Protein Data Bank
accession numbers 2BXG, 1PW6 and 1V9E).
We synthesized LNA conjugates, starting from amino-tagged
locked nucleic acid precursors, bearing a primary amino group
with a C6 spacer. The LNA strand, which was modified at the 5’
extremity, also carried a fluorescein moiety on the last base,
facilitating affinity measurements in solution using fluorescence
polarization.19,20 In physiological buffer conditions, the
incorporation of a 2-hydroxybenzaldehyde moiety (11-LNA) on
the complementary LNA strand enhanced the affinity of 4-(4-
iodophenyl)butanoic acid to HSA by 5-fold, compared to
benzaldehyde (1-LNA) or phenyl (0-LNA) derivatives [Figure 3].
Figure 3. Affinity measurements of protein ligands using fluorescence
polarization methodologies and LNA derivatives. An affinity gain for LNA
derivatives featuring the reactive 11-LNA moiety was observed for HSA and
IL2, but not for CAII. The affinity gain could be abolished by incubation with a
molar excess of hydroxylamine. FA = Fluorescence Anisotropy
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(BM-Ac), as well as the BM-Ac-0 and BM-Ac-1 compounds,
used as negative controls. [Figure 4].
The concept of using a bidentate binding mode in order to obtain
a
high-affinity protein recognition has previously been
investigated both for small organic ligands and for recombinant
antibodies.23-24 The simultaneous engagement of two binding
sites has been exploited in dynamic ligation screening, forming
covalent bonds between synergistic fragments in the presence
of a target protein of interest.25 Large compound collections,
displayed at the extremities of complementary DNA strands
which can combinatorially self-assemble, can be screened using
dual-pharmacophore encoded chemical library technology.26
DNA-encoded libraries have previously been used to discover
irreversible covalent inhibitors.27 Similar procedures could be
implemented for the discovery of reversible covalent binders, by
incorporating 2-hydroxybenzaldehyde or similar moieties during
library construction. Encoded self-assembling chemical (ESAC)
libraries are particularly attractive for the discovery of covalent
inhibitors, since DNA conjugates can be individually purified to
homogeneity, thus preserving the integrity of reactive functional
groups.11,19 Moreover, DNA structures have been used to probe
the magnitude of affinity gain by polydentate engagement with
multivalent protein target.28
It will be interesting to investigate whether more reactive lysine
modifiers (or modifiers for other amino acids) can be used for
pharmaceutical applications. From a thermodynamic viewpoint,
practical applications would be limited by the molar
concentration of amino acid residues in serum and in tissues.
However, since Schiff base formation is a relatively slow
reaction, it may be conceivable that a “dock-and-lock” procedure
may allow the selective in vivo modification of target proteins, if
kinetic parameters are judiciously chosen. N-hydroxysuccinimido
derivatives of aptamers have recently been used to promote a
site-specific protein modification in cell cultures.29 It is therefore
Figure 4. Enzymatic inhibition assay for benzamidine derivative against
urokinase-type plasminogen activator (uPa). The space-fill representation of
the three-dimensional structure of the protein (Brookhaven Protein Data Bank
accession numbers 3MWI) reveals an ε-amino group of a lysine residue in
close proximity of the active site (depicted in blue). In an inhibition assay in
possible
that
moieties
more
reactive
than
2-
hydroxybenzaldehyde may be compatible with pharmaceutical
and chemical biology applications.
presence of Z-GGR-AMC as
a fluorogenic substrate, four benzamidine
derivatives were tested. Only the compound bearing the 2-
hydroxybenzaldehyde moiety (BM-Ac-11) showed a 20-fold improvement of
the inhibition capacity.
Notes
The generation of reversible covalent protein binders through
the incorporation of 2-hydroxybenzaldehyde moieties is
potentially applicable to various small molecule ligands. We had
previously shown that the substitution of an LNA moiety with
suitable chemical linkers can lead to an additional affinity gain.21
With this aim, we studied the possibility to generate small
benzamidine derivatives as urokinase-type plasminogen
activator (uPa) inhibitors. uPa over-expression in breast cancer
correlates with poor prognosis and benzamidine (a broad
spectrum serine protease inhibitor) displays a rather modest
inhibitory activity against uPa [180 µM KD].22
D.N. is a co-founder and shareholder of Philogen (www.philogen.com), a
Swiss-Italian Biotech company that operates in the field of DNA-Encoded
Chemical Libraries. J.S. is
(www.philochem.ch).
a board member of Philochem AG
Acknowledgements
The authors gratefully acknowledge financial support from ETH Zürich,
the Swiss National Science Foundation (Project Nr. 310030B_163479/1
and SINERGIA CRSII2_160699/1), ERC Advanced Grant “Zauberkugel”
(670603) and Kommission für Technologie und Innovation (Grant Nr.
17072.1). D.N. and A.D.C. acknowledge the Novartis Foundation for
medical–biological Research for financial support. Thanks are due to Mr.
N. Favalli for preliminary experiments and fruitful discussions
The ε-amino group of the Lys143 of uPa is in close proximity to
the benzamidine binding site [Figure 4]. We generated four
benzamidine derivatives devoid of the LNA scaffold and studied
their inhibitory activity towards uPA. The molecule containing the
2-hydroxybenzaldehyde moiety showed
a 20-fold improved
potency compared to N-(4-carbamimidoylbenzyl)acetamide
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Keywords: Aldehydes; Covalent inhibitors; DNA; DNA-
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COMMUNICATION
Alberto Dal Corso,# Marco Catalano,#
Anja Schmid, Jörg Scheuermann and
Dario Neri*
Page No. – Page No.
Affinity enhancement of protein
ligands by reversible covalent
modification of neighboring lysine
residues
The simultaneous display of protein ligands and of 2-hydroxybenzaldehyde derivatives on either two complementary LNA strands or
small-organic molecule lead to an enhanced binding affinity by reversible covalent interaction with proximal lysine residues.
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