Platination of cysteine by an epidermal growth factor receptor kinase-targeted hybrid agent

Article abstract of DOI:10.1039/c8cc04251a

Hybrid molecules have been developed which are comprised of a tyrosine kinase-targeted, quinazoline-based scaffold and a flexibly linked dia(m)minechloridoPt(ii) moiety. The target compounds maintain high affinity and selectivity for ErbB family kinase proteins and one of the derivatives induces platinum adducts with a pharmacologically important cysteine residue.

Full text of DOI:10.1039/c8cc04251a

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
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Platination of Cysteine by an Epidermal Growth Factor Receptor
Kinase-Targeted Hybrid Agent†
Mu Yang,a Hanzhi Wu,b Julie Chu,a Lucas A. Gabriel,a Karen S. Anderson,c Cristina M. Furduib and
Ulrich Bierbach*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Hybrid molecules have been developed which are comprised of a formation via Michael addition chemistry between a suitably
tyrosine kinase-targeted, quinazoline-based scaffold and a flexibly modified group on the inhibitor and a noncatalytic cysteine
linked dia(m)minechloridoPt(II) moiety. The target compounds residue near the ATP binding pocket.10, 11 This strategy has
maintain high affinity and selectivity for ErbB family kinase proteins proven successful in cancers that have not acquired resistance
and one of the derivatives induces platinum adducts with a due to EGFR-independent bypass signaling pathways.12
pharmacologically important cysteine residue.
We have recently begun to explore the use of precious
metals as electrophilic “warheads” in molecularly targeted
therapies.13, 14 In the present study, we wanted to answer the
question whether a monofunctional Pt(II) moiety could be
introduced into a TKI as a thiol-reactive metalloelectrophile and
if such a compound would be capable of inducing permanent
adducts within the enzyme’s active site. Here, we demonstrate
that the modification of a TKI with platinum is compatible with
EGFR kinase–inhibitor recognition and provide mass
spectrometric evidence for adduct formation with cysteine in a
model peptide and in EGFR kinase.
Activating mutations within the protein kinase complement of
the human genome (kinome) are responsible for a large number
of diseases, including cancer.1-3 Because of their favorable
toxicity profiles and cancer tissue-specific mechanism of action,
protein kinase inhibitors have become a major class of
personalized oncology drugs.4 Tyrosine kinases belonging to
the epidermal growth factor receptor family (ErbB, e.g. EGFR
and Her2) are a major target for treating patients diagnosed
with tumors harboring mutationally activated or overexpressed
oncogenes.5 One promising oncology drug, gefitinib (Fig. 1a), a
(b)
4-(3’-chloro-4’-fluoroanilino)quinazoline derivative, is
a
molecularly targeted therapy indicated for the management of
EGFR-positive lung adenocarcinomas and other solid tumors.6
Tyrosine kinase inhibitors (TKIs) such as gefitinib have been
designed to outcompete adenosine triphosphate (ATP), the
enzymes’ natural substrate, for active site binding, thereby
suppressing phosphorylation signals that lead to uncontrolled
8
cell proliferation and cancer progression.7, Unfortunately,
secondary active site mutations often diminish the therapeutic
efficacy of gefitinib and other reversible kinase inhibitors.9 This
obstacle has recently been addressed by designing TKIs into
irreversible binders to block ATP binding more effectively.
These derivatives most commonly employ covalent bond
a.Department of Chemistry, Wake Forest University, Wake Downtown Campus,
Winston-Salem, NC 27101, USA. Email: bierbau@wfu.edu
b.Department of Internal Medicine, Section on Molecular Medicine, Wake Forest
School of Medicine, Winston-Salem, NC 27157, USA
Fig. 1 (a) Structures of gefitinib and the newly designed platinum–TKI hybrid agents 1–4
(tmeda stands for N,N,N’,N’-tetramethylethylenediamine), and (b) proposed mechanism
of the hybrid agents involving reversible, high-affinity recognition of the ATP-binding
pocket and subsequent irreversible cysteine modification.
c. Department of Pharmacology, Yale University School of Medicine, New Haven, CT
06520, USA
† Electronic Supplementary Information (ESI) available: Experimental procedures,
compound characterization, enzyme assays, and mass spectrometry data. See
DOI: 10.1039/x0xx00000x
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The quinazoline-derived pharmacophore tolerates a wide range family kinases, except for ErbB3, which show relatively weak
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DOI: 10.1039/C8CC04251A
of structural modifications of the side chain at C6 of the binding (81% of control). Wild-type EGFR kinase and several of
quinazoline C6 ring, which protrudes out of the ATP binding its mutated forms show complete displacement of surrogate
15
pocket of the EGFR kinase domain.13,
Installation of a (0% of control), which corresponds to low-nanomolar Kd
platinum(II) moiety in that location positions the metal in values.17 The only non-ErbB targets mapped in this assay are
proximity to EGFR-C797 in the hinge region of the kinase FMS-related tyrosine kinase (FLT3) and lymphocyte-specific
domain, in complete analogy with the design of Michael protein tyrosine kinase (LCK). LCK has previously also been
acceptor-modified TKIs.11
In the monofunctional identified as a target of gefitinib.17 Overall, the selectivity
closely mimics that reported
[PtCl(diam(m)ine)]+ moieties introduced in this study, the profile established for compound
1
chlorido ligand serves as a leaving group to promote ligand for gefitinib.17 These findings indicate that modification of the
exchange with cysteine sulfur. Based on previous docking quinazoline scaffold at the C6 position with a monofunctional
experiments,13 this design should favor formation of a platinum- Pt(II) moiety via a flexible linker (X-(CH2)2-N, X = NH or O) does
mediated TKI–enzyme cross-link (Fig. 1b).
The target not affect the reversible binding step of the quinazoline ligand
compounds in this study were generated by reacting a terminal with the ATP-binding pocket. This outcome is consistent with
secondary amino group installed on the C6 side chain of a solid state structures of EGFR kinase–inhibitor complexes15 and
quinazoline derivative13 with a platinum–nitrile complex to our previous findings using protein–ligand modeling.13 These
produce an amidine linkage16 (Fig. 1a, compounds
1–3). studies demonstrated that the extended side chain at
Another derivative was generated via direct replacement of a quinazoline-C6 is located at the entrance of the ATP-binding
chlorido ligand with amine nitrogen in a silver ion-activated cis- pocket and is not involved in specific interactions with the active
platinum precursor (Fig. 1a, compound
synthetic schemes and procedures). In prototypes
side chain on C6 is connected to the quinazoline ring via an
amino group. In compounds and the NH-group was replaced
4
) (see the ESI for site that might be disrupted by platinum and compromise TKI–
1
and , the kinase recognition.
2
3
4
with an ether linkage, which improved the stability of the
hybrids. Monodentate NH3 and bidentate amine ligands were
introduced as nonleaving groups to study the effects of ligand
sterics on receptor–ligand interactions.
Because the new hybrid agents were derived from
gefitinib’s core scaffold, the design promises strong binding to
EGFR kinase. An ATP-independent competitive binding assay17
was performed with compounds
1 and 2 to determine if the
newly introduced cationic Pt(II) moieties affect kinase binding
affinity and selectivity (reversible binding step in Fig. 1b).
Compounds
type EGFR of 1.0 nM and 3.4 nM, respectively (see the ESI for
dose–response curves and details of the assay). For
1 and 2 showed dissociation constants (Kd) in wild-
comparison, the Kd reported for gefitinib determined by the
same assay is 0.9 nM.17 These results suggest that installation
of platinum does not compromise the nanomolar binding
affinity of the quinazoline pharmacophore. Replacement of NH3
(in
(in
1
2
) with the sterically more hindered bidentate tmeda ligand
) leads only to a minor decrease in binding affinity,
demonstrating that the binding site tolerates major structural
variations within the platinum coordination sphere.
Using the same assay, compound
1 was also screened
against a panel of 145 kinase domains representing the human
protein kinase “cysteinome”, a group of approximately 200
kinases containing targetable active-site cysteines.18, 19 These
include the ErbB family kinases EGFR (wild-type enzyme and 11
oncogenic mutants), ErbB2 (Her2), ErbB3, and ErbB4.
Fig. 2 Competitive kinase binding assay for compound 1 in 145 protein kinases of the
human cysteinome, including several of their mutated forms. Relative binding affinities
are represented by red and green circles in a phylogenetic kinome tree for wild-type
enzymes (top) and mutant variants (bottom). A larger red circle indicates a higher
binding affinity. A total of 17 interactions with binding levels of less than 35% of control
(red circles) at a fixed concentration of test compound of 1 M were mapped. Selectivity
factor, S35 = 0.052. See the ESI for assay details and complete binding data (Table S3).
Compound
1 showed a high binding affinity for 17 of the 145
tested kinases, based on its ability to displace more than 65% of
kinase from an immobilized substrate surrogate (reported as
the percentage of kinase that remains bound).17 High-affinity
targets are highlighted as red circles in a phylogenetic tree in
Fig. 2 (see the caption and the ESI for details). The highest
affinities among the top 17 kinases were observed for ErbB
The results from the kinome screening experiments
unequivocally demonstrate that the platinum moieties in the
respective hybrid agents have no major effect on the binding
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affinity and selectivity of the quinazoline scaffold. Likewise, investigated. Tryptic digests were analyzed byViHewRA-rMticlSe Oanlninde
cell-free kinase assays performed in the presence of ATP to tandem MS to sequence the peptides aDnOdI:l1o0c.1a0t3e9/tCh8eCsCi0te42s5o1Af
study substrate phosphorylation demonstrated that our hybrids modification. (To eliminate the possibility of nonspecific
are as efficient at inhibiting kinase function as gefitinib (see the reactions with the digested peptides and ensure that only
ESI). The above assays are designed to measure equilibrium adducts formed in the intact protein are detected, excess hybrid
binding and do not provide insight into the proposed reactivity agent was removed prior to proteolytic digestion.) Masses
of platinum in the hybrids with kinase cysteine (Fig. 1b, (m/z) representing both the monofunctional adducts and
irreversible binding step).
To detect and structurally chelates in A1–A4 were included as possible modifications
characterize potential cysteine modifications, reactions were when searching for platinum-containing peptides.
monitored by in-line high performance liquid chromatography–
high resolution mass spectrometry (LC–HRMS) in conjunction
with tandem mass spectrometry (MS/MS). Prior to experiments
with human recombinant EGFR kinase, the reactivity of two
representative compounds,
3
and
4
,
with
a synthetic
octapeptide, QLMPFGCL, was studied.
This sequence
represents amino acid residues 791–798 in the hinge region of
the EGFR kinase domain, including the proposed target, C797.
LC–MS analysis of the mixtures resulting from incubations of
the octapeptide with compounds 3 and 4 indicate that platinum
in both cases reacts with C797 in two distinct ways.
Furthermore, tandem mass spectroscopy was used to confirm
the amino acid and sequence selectivity of the adducts formed.
For both compounds, the data is consistent with a mechanism
by which cysteine sulfur replaces the chloride leaving group.
This results in monofunctional adducts in which platinum
produces a cross-link between the quinazoline ligands and the
peptide (adducts A1 and A3, Fig. 3A,B; for LC–MS traces and
product analysis, see the ESI). In addition, a second ligand
exchange occurs for both hybrid agents. In compound 3, one of
the two ammine (NH3) ligands is lost, resulting in a bifunctional
S,N-chelate involving cysteine sulfur and deprotonated amide
nitrogen of the peptide backbone, a common motif previously
observed in cis-platinum–peptide adducts (adduct A2, Figure
Fig. 3 Reaction of compounds 3 (A) and 4 (B) with the target peptide QLMPFGCL based
on product analysis by LC–MS. T2´ and T2 stand for the amidine-linked and amine-linked
quinazoline ligand (X = O), respectively. (C) Characterization of adduct A4 by MS/MS,
confirming the structure and the sequence specificity of a [Pt(pn)]2+ chelate (inset).
Selected fragment ions are labeled; see the ESI for a complete list of b and y ions.
3A).20-22 A similar situation is observed for compound
4. In this
case the reaction results in the substitution of the quinazoline
ligand (T2), which becomes a leaving group, while the propane-
1,3-diamine (pn) chelate remains intact. The result of this
reactivity is the transfer of the [Pt(pn)]2+ moiety from the
quinazoline ligand to C797, yielding adduct A4 (Fig. 3B).
The data generated for compound
4 confirms that the
[Pt(pn)]2+ chelate observed in model adduct A4 also forms
within the native enzyme. By contrast, no monofunctional
adducts were detected in which the quinazoline moiety
remained cross-linked to the protein (see adduct A3). Three
peptides were identified in which platinum chelated one of the
following cysteine residues: C797, C939, and C781 (Fig. 4; see
ESI for MS/MS data). Platination of C797, the pharmacologically
relevant of the six cysteine residues in EGFR kinase, supports
the proposed mechanism of targeted delivery of platinum into
the ATP-binding hinge region of the enzyme. Because
incubations were performed with excess hybrid agent,
formation of additional (nonspecific) adducts is also observed
with C939 in the C-terminal lobe and C781 in the N-terminal
lobe of the kinase domain. These residues are located at the
protein surface but exhibit more limited solvent accessibility
than C797 (Fig. 4). No adducts were detected for the deeply
buried cysteine residues. Surprisingly, none of the adducts
When tandem MS was performed on A1, A2, and A3 to
confirm the structures and sequence specificity of the adducts,
fragmentation of the modified sequence by higher-energy
collisional dissociation (HCD) also led to the dissociation of
platinum from the peptide. By contrast, the bis-chelated
platinum adduct A4 remained intact, and several relevant
platinum-containing b and y ions (see the ESI) were observed
(Fig. 3C). The results unequivocally demonstrate that platinum
selectively targets cysteine. Adduct formation with methionine
sulfur (M793) in this sequence, which is also known to bind
platinum(II) with high affinity,20 is not observed. Finally, based
on the pattern of platinum-modified b and y ions, the chelate
forms with sulfur and amide nitrogen of cysteine and bidentate
coordination involving a non-cysteine nucleophile is unlikely.
To determine if any of the adducts identified in the synthetic
octapeptide (A1
–A4) also form in the intact enzyme, the
reactions of compounds
3
and with the 88-kDa truncated
4
formed by compound
3
in the model peptide were observed in
intracellular kinase domain of human recombinant EGFR were
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EGFR kinase. One reason for this outcome may be that platinum
in is positioned within the binding pocket in a way that
Conflicts of interest
There are no conflicts to declare.
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DOI: 10.1039/C8CC04251A
3
disfavors binding to C797. In addition to undetectable levels of
modification, it is also possible that any adducts formed by this
agent may be too labile and susceptible to collision-induced
dissociation, as demonstrated for this analogue in the model
octapeptide.
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Fig. 4 Summary of cysteine adducts identified in EGFR tyrosine kinase based on tandem
mass spectrometry analysis of tryptic digests of enzyme treated with compound 4. Pt-
modified cysteine residues are highlighted in red. [Pt(pn)]2+ is represented by an asterisk.
The model octapeptide sequence, Q791–L798, in the hinge region between the N- and
C-lobes, is highlighted in magenta. Structure adopted from PDB ID: 3W2S.
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that is compatible with kinase active site recognition. In
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unique mechanism that leads to the formation of a platinum(II)
chelate at C797. This form of TKI-mediated delivery of a
metalloelectrophile has potential applications in modulating
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ATP-driven kinase signaling, which contrasts other metal-based
23
hybrid designs.14,
Kinase cysteine is not only a
pharmacologically relevant target for irreversible inhibitors.
Cysteine modifications under oxidative stress conditions, for
instance, have also been implicated in kinase regulation.24, 25
Similarly, platination of this site by our hybrid agents may also
lead to conformational changes affecting protein
phosphorylation and signal transduction. In conclusion, the
unique reactivity and cysteine modifications observed for the
platinum-modified TKIs suggest a potential new role of 21 A. K. Boal and A. C. Rosenzweig, J. Am. Chem. Soc., 2009, 131
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platinum, the classical anticancer metal, as an electrophile in
molecularly targeted drugs. The results of this study warrant
future experiments to delineate structure–function
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work was funded by the Wake Forest Innovation and
Commercialization Services and the Wake Forest University
Comprehensive Cancer Center (NCI CCSG P30CA012197).
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TOC
Platinum-modified tyrosine kinase inhibitors show strong and selective EGFR kinase binding and form
adducts with a pharmacologically relevant cysteine residue.