Virtual Screening Identifies Irreversible FMS-like Tyrosine Kinase 3 Inhibitors with Activity toward Resistance-Conferring Mutations

Article abstract of DOI:10.1021/acs.jmedchem.8b01714

The use of covalent irreversible binding inhibitors is an established concept for drug development. Usually, the discovery of new irreversible kinase inhibitors occurs serendipitously, showing that efficient rational approaches for the rapid discovery of new drugs are needed. Herein, we report a virtual screening strategy that led to the discovery of irreversible inhibitors of FMS-like tyrosine kinase 3 (FLT3) involved in the pathogenesis of acute myeloid leukemia. A virtual screening library was designed to target the highly conserved Cys828 residue preceding the DFG motif by modification of reported reversible inhibitors with chemically reactive groups. Prospective covalent docking allowed the identification of two lead series, resulting in a massive increase in inhibition of kinase activity and cell viability by irreversible inhibitors compared to the corresponding reversible scaffolds. Lead compound 4b (BSc5371) displays superior cytotoxicity in FLT3-dependent cell lines to compounds in recent clinical trials and overcomes drug-resistant mutations.

Full text of DOI:10.1021/acs.jmedchem.8b01714

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Article
Virtual Screening Identifies Irreversible FMS-like Tyrosine Kinase
Inhibitors with Activity towards Resistance-conferring Mutations
3
Dennis Bensinger, Daniel Stubba, Anjali Cremer, Vanessa Kohl, Theresa Waßmer, Johanna
Stuckert, Victoria Engemann, Kimberly Stegmaier, Katja Schmitz, and Boris Schmidt
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01714 • Publication Date (Web): 11 Feb 2019
Downloaded from http://pubs.acs.org on February 12, 2019
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Virtual Screening Identifies Irreversible FMS-like
Tyrosine Kinase 3 Inhibitors with Activity towards
Resistance-conferring Mutations
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†
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Dennis Bensinger , Daniel Stubba , Anjali Cremer , Vanessa Kohl , Theresa Waßmer ,
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Johanna Stuckert , Victoria Engemann , Kimberly Stegmaier , Katja Schmitz and Boris
Schmidt†*
†Clemens-Schöpf-Institute for Organic Chemistry and Biochemistry, Technische Universität
Darmstadt, 64287 Darmstadt, Germany
‡Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston,
Massachusetts 02215, United States
Abstract
The use of covalent irreversible binding inhibitors is an established concept for drug
development. Usually the discovery of new irreversible kinase inhibitors occurs
serendipitously showing that efficient rational approaches for the rapid discovery of new
drugs are needed. Herein, we report a virtual screening strategy that led to the discovery of
irreversible inhibitors of the FMS-like tyrosine kinase 3 (FLT3) involved in the pathogenesis
of acute myeloid leukemia (AML). A virtual screening library was designed to target the
highly conserved Cys828 residue preceding the DFG motif by modification of reported
reversible inhibitors with chemically reactive groups. Prospective covalent docking allowed
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the identification of two lead series, resulting in a massive increase in inhibition of kinase
activity and cell viability by irreversible inhibitors compared to the corresponding reversible
scaffolds. Lead compound 4b (BSc5371) displays superior cytotoxicity in FLT3-dependent cell
lines to compounds in recent clinical trials and overcomes drug-resistant mutations.
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Introduction
Acute myeloid leukemia (AML) is a malignant neoplasia affecting hematopoietic
progenitor cells. These cells fail to differentiate into functional blood cells of the myeloid
path (erythrocytes, neutrophils and thrombocytes), while ongoing cell proliferation leads to
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the accumulation of myeloblasts in the bone marrow. The most common pathogenic
mutations occur on the gene encoding for FMS-like tyrosine kinase 3 (FLT3). Length
mutations in this gene through internal tandem duplications (ITD) in the juxtamembrane
domain occur in about 25% of patients and lead to ligand-independent auto-phosphorylation
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and downstream signaling. Point mutations in the FLT3 kinase domain (e.g. D835Y) occur
in 10% of patients and destabilize the inactive kinase conformation leading to higher kinase
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activity. In 2017 the multitargeted kinase inhibitor Midostaurin (PKC412) was approved by
the US Food and Drug Administration (FDA) in combination with chemotherapy for the
treatment of de novo FLT3-mutated AML showing a 22% reduction of risk of death.4
Minimal residual disease after treatment with tyrosine kinase inhibitors (TKIs) like sorafenib
and quizartinib can lead to relapse and is often associated with resistance-conferring point
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mutations in an FLT3-ITD allele. As emergence of drug-resistant mutations is a common
liability of targeted therapies, new agents with distinct properties are needed. The use of
irreversibly binding TKIs has several advantages over classical reversible inhibitors such as
an increased target residence time, altered selectivity, as well as more durable responses than
their reversibly binding counterparts while idiosyncratic toxicity and sustained off-target
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effects are additional risks. They act by the formation of a covalent bond between an amino
acid with a nucleophilic side chain such as cysteine and a chemically reactive electrophilic
group (CRG) connected to a specific ligand. Bond formation usually takes place after a strong
non-covalent complex has formed that positions the CRG in close vicinity to the target
nucleophile. Upon covalent bond formation, irreversible reactions rule out ligand
dissociation leading to non-equilibrium inhibition kinetics. Thus, the enzyme function can
be restored by de novo synthesis only.
So long, four TKIs are approved by the FDA for the treatment of certain malignancies,
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afatinib, neratinib, osimertinib, and ibrutinib, act by irreversibly inhibiting the EGF-
receptor family and Bruton´s tyrosine kinase, respectively.
The natural product hypothemycin is a resorcylic acid lactone that irreversibly inhibits 18
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kinases, including FLT3. The cis-enone motif is attacked by a cysteine directly preceding
the DFG motif at the start of the activation loop (Cys828 in FLT3). These so-called “group 4”
cysteines are the most conserved in the human kinome occurring in 48 of about 500
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kinases. A crystal structure analysis of hypothemycin and ERK2 confirms covalent binding
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in the DFG-in conformation. Despite the advantages of irreversible inhibitors, only few
drug development efforts have focused on targeting this group of cysteines. Examples include
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benzoquinone-substituted quinazolines targeting VEGFR2 and analogues or ring-opened
derivatives of resorcylic acid lactones targeting FLT3, showing only micromolar cellular
activity.14
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Recently, discovery of covalent binding inhibitors was advanced by the development of
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molecular modelling algorithms that allow covalent docking. In 2015, our group introduced
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the covalent docking tool DOCKTITE as a freely available script package for the Molecular
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Operating Environment. In this method a CRG is tagged by a pre- or user-defined trans-
formation, that is then attached to a selected covalent attachment point of the receptor.
During docking the nucleophilic residue is detached from the receptor but fixed by a tight
pharmacophore. This strategy allows highly customizable virtual screening strategies while
all docking steps can be monitored and controlled. Consequently, DOCKTITE has been
successfully used for the development of covalent inhibitors for the cysteine proteases
rhodesain,18-19 cruzain and falcipain as well as for the plasmodium proteasome. Despite
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the recent progress described, only one case study identifying covalent kinase inhibitors in
silico has been reported so far. London et al. successfully used the DOCKovalent tool to
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identify covalent reversible inhibitors of the kinases RSK2 and JAK3 as well as irreversible
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MKK7 inhibitors, that were published while this study was under revision. However, the
scope of covalent docking methodology has to be enlarged, as only cyano acrylamides and
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acrylamides were evaluated and hit identification profited by available protein-ligand
structures with the modified target cysteine (RSK2) or numerous cocrystal structures with
related hinge binders (JAK3, MKK7), respectively.
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Because of the impressive clinical benefits of irreversible inhibitors and the vulnerability of
FLT3 treatment to resistance we sought to rationally design and evaluate irreversible FLT3
inhibitors.
In the following, we present a strategy that allows the identification of irreversible FLT3
inhibitors with superior cytotoxicity to inhibitors in clinical trials that can overcome
resistance-conferring mutations of FLT3.
Results and Discussion
Rational Design of Irreversible FLT3 Inhibitors. A major drawback of FLT3 inhibitors in
recent clinical trials was the acquisition of point mutations within the ATP-binding site thus
we decided to target FLT3 in its catalytically active DFG-in conformation. Within the ATP-
binding site FLT3 possesses three targetable cysteine residues. Two of them, Cys694
(gatekeeper + 3) and Cys695 (gatekeeper + 4), are part of the hinge region while Cys828 is
preceding the conserved DFG-motif. Kinase inhibitors usually gain potency by forming
alternating hydrogen bond donor/acceptor interactions with the hinge backbone atoms.
Therefore modification of known hinge binding motifs are promising starting points to target
the neighboring Cys828 residue. Additionally, exposure of Cys828 to the surface of the ATP
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binding pocket allows variation of the trajectory by which a CRG is recognized and a
covalent bond can be formed. However, no crystal structure of FLT3 in the active
conformation has been reported, so we designed a homology model of the active FLT3
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conformation using c-KIT (pdb 1PKG) as a template (Figure S1-S3).24
Figure 1. (a) Virtual screening and filter strategy for the in silico discovery of irreversible
FLT3 inhibitors; (b, c) Structure of CRGs and pose distribution among scaffolds. HA: α-halo
acetamide, AC: acrylamide, VS: vinyl sulfonamide and PA: propargyl amide (d) Rational
design and docking hits of the bisaminopyrimidine scaffold 3 and (e) the indolinone inhibitor
scaffold 6 (gk = gatekeeper residue).
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c-KIT is the most closely related kinase to FLT3 with a sequence similarity of 79% and a
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sequence identity of 64% and consequently numerous shared potent inhibitors. To validate
the homology model, we docked hypothemycin covalently to Cys828 of FLT3. Rigid body
alignment of the top-scored conformation docked into FLT3 with the conformation obtained
from the co-crystallized structure in ERK2 gave a r.m.s. deviation (RMSD) of 1.34 Å, while
the top-scored redocked pose in ERK2 showed a RMSD of 1.08 Å (Figure S4). To identify
potent reversible inhibitors as starting points for reactive modifications and subsequent
covalent docking, the publicly accessible BindingDB Database was manually searched for
FLT3 inhibitors with distinct hinge binding motifs (Figure 1a, 1752 entries as of Jan. 2015).26
Typical inhibitors that were excluded contain bisarylurea motifs such as sorafenib,
quizartinib and ponatinib that are designed to occupy the hydrophobic pocket II and thus
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bind to the inactive DFG-out conformation. We found a set of 13 different hinge binding
scaffolds (Figure S5) and identified a plausible reversible binding pose for each scaffold
(
Figure S6 – S18). Those scaffolds form mono- (5 scaffolds), bi- (6 scaffolds) or tridentate (2
scaffolds) alternating hydrogen bond donor/acceptor interactions to the gatekeeper+1
(Glu692) and +3 residue (Cys694) that are comparable to hinge binding observed from crystal
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structures of related scaffolds. We selected four chemically and sterically distinct CRGs
(
Figure 1b) to be attached to the non-covalent docked structure. The acrylamide (AC), vinyl
sulfonamide (VS) and propargyl amide (PA) groups span approximately 5 Å, while the α-halo
acetamide (HA) warhead spans only 3.8 Å (Figure S19). We concluded, that CRG-
substitution of aromatic ring carbons within 5 Å to the Cys828 sulfur may enable covalent
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binding (Figure S6F to S18F), while favorable conformations were selected by covalent
docking. Subsequently, we identified 32 attachment points among the selected scaffolds
leading to a targeted virtual library of 128 compounds (Table S1). Since addition of
nucleophiles to the propargyl amide can lead to E- or Z- substituted alkenes, addition to the
covalent attachment point prior to pharmacophore docking gave 160 docking input
structures.
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Virtual Screening and Filter Strategy. Covalent docking was performed using the
pharmacophore placement method. The pharmacophore features of the 13 scaffolds were
defined by the aryl, donor and acceptor groups forming hinge interactions and
complemented with a tight pharmacophore at the target cysteine (Figure S6E to S18E).
However, the pharmacophore feature of the nucleophilic sulfur atom in Cys828 was less
stringent (d = 3.0 Å) allowing different rotamers. The initial docking gave 831 poses (Table
S2, Figure S20) that were filtered for Glu692 and Cys694 backbone hydrogen bond donor
acceptor interactions (404 poses left) and subsequently rescored using the DSX scoring
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function while only the top 5 poses of every ligand were retained (319 poses left). We then
calculated the ligand efficiency based on the London dG score and set a cutoff at -0.5
kcal/mol/heavy atom to limit the search to highly efficient inhibitors (102 poses left, Figure
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c, Table S3 – S5). From the reversible scaffolds, 5 scaffolds retain more than 10 hits of the
corresponding reactive inhibitors (73% of total hits). On average, 32% of the initial docking
hits in this group ranging from 13% (bisaminopyrimidine, scaffold 3) to 67% (indolinone,
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scaffold 6) are retained by the filter strategy. The hit distribution varies widely among the
CRGs. With 45% of total hits vinyl sulfonamides are the most abundant group, followed by
the α-halo acetamides (29%), acrylamides (16%) and propargyl amides (10%).
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Characterization of Reversible Binding Fragments. As the potency of irreversible binding
inhibitors is based on a moderate affinity between target and ligand in a reversible
equilibrium prior to inactivation, we sought to evaluate the potency and structure-activity
relationship of the selected reversible hits to further increase efficiency and the success rate
of prospective irreversible inhibitor design. As the rational design starts from reported
reversible ligands, synthetic procedures used to prepare reversible compounds 1a to 1g were
based on earlier work (see experimental section). Among the initial series of reversible
inhibitors (1a to 1e), bisaminopyrimidine 1a is the most cytotoxic compound (89% cell death,
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0 µM, Table 1) and strongest inhibitor of FLT3wt. The indolinone 1b shows a comparable
inhibition for FLT3wt and FLT3(D835Y). Imidazopyridine 1c and quinazoline 1d are less
potent inhibitors of FLT3(D835Y) while azaindole 1e shows only weak inhibition of FLT3wt.
However, all of the compounds of the initial series show only low cytotoxic activity in FLT3-
ITD mutant MV4-11 cells. To increase reversible binding affinity, we applied small changes
to the most promising inhibitors 1a and 1b that are compatible with the initial, reversible
docking poses leaving the attachment points for the CRGs unchanged (Figure 1d, e; S21a, b).
Exchange of the phenyl ring in 1b with a 2-pyrrole (1g) constrains a planar conformation of
the ligand by formation of an intramolecular hydrogen bond. The 5-methyl group connected
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to the pyrazolyl amine 1a was changed to a cyclopropyl ring (1f) that has also been reported
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to efficiently target FLT3. These small modifications massively increase the cytotoxicity of
both scaffolds as well as inhibition of FLT3 and FLT3(D835Y) kinase activity making these
fragments an efficient starting point for the development of irreversible inhibitors. The same
modifications were applied to the poses obtained from the initial covalent docking and
subsequent energy-minimization leaves the poses largely unchanged (Figure S21c – j).
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Table 1. Inhibition of FLT3wt; FLT3(D835Y) and MV4-11 cell viability treated with
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b
reversible binding fragments. Eurofins KinaseProfiler, ADP-Glo Assay.
IC50
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4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Relative Inhibition (cTKI = 1 µM) / %
Structure
Compound
(MV4-11)
/ nM
FLT3wt a
FLT3 (D835Y)b
H
N
N
NH
N
N
1
a
> 1.000 nM
82
15
HN
H
O
N
1
b
> 1.000 nM
> 1.000 nM
60
76
45
8
N
1
c
N
O
N
N
O
1
d
> 1.000 nM
> 1.000 nM
26
25
25
83
HN
H
N
N
1e
H
N
N
NH
N
N
1
f
230±53
380±21
98
97
88
86
HN
H
O
N
NH
1
g
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Characterization of Indolinone-based Irreversible Inhibitors. A total of nine inhibitors based
on the reversible indolinone hit 1g were synthesized (Table 2). In addition to the CRG-
substituted derivatives we synthesized the analogous unreactive inhibitors to assess the effect
of the amide or sulfonamide linking group on reversible binding affinity. To evaluate the hit
rate of the screening strategy, we also synthesized the inhibitors that were not identified as a
hit during virtual screening. The 5-substituted vinyl sulfonamide 2i was the most potent
inhibitor of this series showing a 5-fold in-crease in inhibition of MV4-11 cell viability
compared to the initial fragment as well as the highest inhibition of kinase activity in
FLT3(D835Y) and FLT3(ITD) in this series. Importantly, the unreactive ethyl sulfonamide 2h
shows a massive decrease in inhibition of cell viability and kinase activity with decreased
inhibition in FLT3(D835Y) and FLT3(ITD) suggesting that potency arises from covalent bond
formation as both compounds are able to adopt the same reversible conformations. The vinyl
sulfonamide warhead gave the highest number of hits in silico with the top virtual ligand-
efficiency (LE ) at rank 6 of the 102 hits (LE = -0.55) and further poses on ranks 16, 22, 41
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
V
V
and 67. The propargyl amide 2g as well as acrylamide 2f shows a massive loss of cytotoxic
activity compared to the initial hit similar to the unreactive propione amide 2e and low
inhibition in kinase assays.
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Table 2. Inhibition of cell viability of MV4-11 cells and FLT3-mutant kinase activity by the
indolinone fragment 1g and the corresponding 5-substituted indolinones 2a – i with
chemically reactive electrophilic groups and the analogous unreactive groups.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Relative Inhibition / %
N
H
O
IC50
R
FLT3(D835Y)
Compound
(MV4-11)
FLT3(ITD)
c = 0.1 µM
N
H
/
nM
c = 0.1 µM
c = 1 µM
R =
- H
1g
2a
380 ± 21
> 1.000
40
47
86
90
61
33
O
N
H
O
F
2b
2c
2d
845 ± 20
72 ± 8
57
53
48
92
87
92
51
61
21
N
H
O
Cl
Br
N
H
O
O
653 ± 200
N
H
2
e
> 1.000
15
81
44
N
H
O
O
2
f
> 1.000
709 ± 50
> 1.000
29
5
79
58
72
25
55
9
N
H
2
g
N
H
O
S
2h
2i
13
N
H O
O
S
67 ± 8
77
96
93
N
H O
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Thus, covalent inhibition is not favorable for acrylamide 2f though it had been identified
in a single pose during virtual screening (rank 1, LE = -0.61) obtaining a similar
V
conformation as 2i. The α-halo acetamides show comparable inhibition of kinase activity in
FLT3(D835Y) with the unsubstituted acetamide while a slight preference for the α-chloro
acetamide was found inhibiting FLT3(ITD). While the reversible acetamide-substituted
indolinone fragment 2a shows no inhibition of MV4-11 cell viability, the α-fluoro- (2b) and
α-bromo-substituted (2d) acetamides are less active than the initial screening hit 1g. The α-
chloro acetamide 2c shows similar cytotoxicity as the vinyl sulfonamide 2i. Having
established the structure-activity relationship of indolinone-based irreversible inhibitors we
reanalyzed the results from covalent docking to explain disagreements between predicted
and confirmed hits.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A possible cause for the false-positive hit acrylamide 2f might be the negligence of the
covalent bond formation as only the reversible fragment and the final covalent complex are
modeled during covalent docking. The energy profile of the dihedral angle between the C-C-
N plane of the aromatic amine and the C-N-S plane of the reactive group in the top-scored
pose of the active vinyl sulfonamide 2i shows that the energy is close to a local minimum
while the dihedral angle is only changed by 3° (56° to 59°) after energy-minimization of the
unbound ligand (Figure 2a, S22). The position of the targeted distal carbon atom is slightly
shifted by 0.72 Å giving an overall RMSD of 0.49 Å. In contrast, the position of the distal
carbon atom in the top-scored docking pose of the inactive acrylamide 2f is changed by 2.24
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Å while the dihedral angle is changed by 39° (overall RMSD 0.89 Å) explaining that the
identified hit could not be confirmed experimentally as the modeled conformation of the
CRG is unfavorable (Figure 2b). Furthermore, conformational search of unbound ligands 2f
and 2i shows that the acrylamide warhead is oriented in-plane of the indolinone ring, while
the vinyl sulfonamide retains multiple conformations out-of-plane enabling induced fit to
the ATP binding site and vicinity to Cys828 (Figure S23). We further assume that the initial
pharmacophore distance of 1.5 Å might be too stringent to allow binding of all warheads, as
the α-halo acetamide series was a false negative hit. Consequently, redocking of the α-halo
acetamide derivatives 2b to 2d was performed using 2 Å ligand pharmacophores. This
adjustment led to numerous plausible docking poses and thus showed to be predictive for
covalent inhibition by α-halo acetamides (Figure S24).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Characterization of Bisaminopyrimidine-based Irreversible Inhibitors. Similar to the
indolinone scaffold, we synthesized 14 inhibitors of the 3- and 4-substituted
bisaminopyrimidine scaffold including derivatives with all four types of CRG to include
structures predicted as hits and predicted negatives as well as the corresponding reversible
substitutions (Table 3). Similarly, the vinyl sulfonamide derivatives 3m and 3n of the initial
fragment 1f show massively increased inhibition of MV4-11 cell viability while the
corresponding reversible structures 3k and 3l show similar cytotoxicity as the reversible
fragment 1f. Inhibition of FLT3 activity shows a comparable increase in inhibition for the
reactive derivatives towards FLT3(D835Y) and FLT3(ITD). Interestingly, 3m shows lower
inhibition of FLT3(ITD) but 25-fold increased cytotoxicity in MV4-11 cells suggesting
synergistic off-target effects. The 3-methyl derivatives corresponding to 3m (2 hits, rank 51,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
6) and 3n (3 hits, rank 17, 54, 83) have been identified during virtual screening and
redocking of 3m and 3n gave similar conformations (Figure S21k – n). These results show
that the screening protocol chosen allows predictive hit finding for the bisaminopyrimidine
scaffold as well.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Inhibition of cell viability of MV4-11 cells and FLT3-mutant kinase activity by the
bisaminopyrimidine fragment 1f and the corresponding 3- and 4-substituted inhibitors 3a – n
with chemically reactive electrophilic groups or the analogous unreactive groups.
H
N
Relative Inhibition / %
FLT3(D835Y) FLT3(ITD)
N
NH
N
N
IC50
HN
Compound
1f
Substitution
(MV4-11)
R
/
nM
c = 0.1 µM
c = 1 µM
c = 0.1 µM
R=
-H
-
230 ± 53
> 1.000
52
36
88
85
27
54
O
3
a
3
N
3
b
4
3
4
3
4
3
4
3
4
3
4
3
> 1.000
175 ± 2
45
33
39
37
34
22
29
38
45
32
72
47
86
90
93
90
80
81
84
58
54
92
96
90
82
74
74
66
22
50
59
2
H
O
3c
Cl
N
H
3
d
141 ± 13
> 1.000
O
3
e
N
3f
3g
3h
> 1.000
H
O
O
787 ± 100
521 ± 65
> 1.000
N
H
3
i
N
H
3
j
> 1.000
17
59
86
73
O
S
3
k
489 ± 17
129 ± 16
2.3 ± 0.1
N
3
l
H O
O
3m
S
N
3
n
4
58 ± 16
86
95
99
H O
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The acrylamide substitution shows no benefit in cytotoxicity compared to the reversible
fragment 1f while the corresponding reversible inhibitors 3e and 3f are almost inactive.
During virtual screening the acrylamides have been identified as low-ranked compounds
only (3g: rank 99 of 102, 3h: rank 49, 101). The 3- and 4- substituted propargylamides 3i and
3j are the least active inhibitors of this series displaying only neglectable MV4-11
cytotoxicity. Although the propargyl amide 3j yielded no poses during screening, 3i was
identified (rank 10, 45). Reversible acetamide-substitution of the initial screening hit leads to
a loss of cytotoxicity in 3- (3a) as well as 4-position (3b) while the α-chloro substitutions in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
c and 3d leads to a slightly increased cytotoxicity compared to fragment 1f suggesting a
covalent mode of action. However, inhibition of FLT3(D835Y) and FLT3(ITD) was
comparable to reversible acetamides 3a and 3b, a trend already observed with the indolinone
α-chloro-acetamide 2c, suggesting that kinase screening alone would under-estimate the
overall potential of this CRG. In summary, the screening strategy presented gave a hit rate of
7
3% based on the number of predicted poses (16 correct-positive, 6 false-positive, Figure
S25) where all of the vinyl sulfonamide derivatives (3 hits, 10 poses) are validated.
Design of 2nd Generation Indolinone Inhibitors. As the design of covalent binding inhibitors
was based on fragmentation of larger lead compounds, we aimed to increase FLT3 inhibition
and cytotoxicity by merging the most active irreversible fragments with the parent scaffold.
The bisaminopyrimidine scaffold is less suitable for this purpose as the pyrazolyl amine motif
may lead to promiscuous kinase inhibitors.30
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Exemplary, we tested activity of the reversible inhibitors 1f and 1g on the far-distant
kinases GSK3α and GSK3β yet containing the DFG-1 Cys residue, showing potent inhibition
for 1f (74% and 76% at 1 µM) but only insignificant inhibition by 1g (12% and -6% at 1 µM).
Furthermore, inhibition of cell viability is about 10-fold higher for the meta substituted vinyl
sulfonamide 3m than for the isomer 3n while inhibition of FLT3 kinase activity shows the
opposite trend suggesting that cytotoxicity may arise from synergistic effects by off-target
inhibition. Furthermore, 3m and 3n are potently cytotoxic towards FLT3wt THP1 cells and
FLT3-negative Jurkat cells (Table 4). Additionally, ligand efficiency in MV4-11 cells is higher
for the indolinone derivatives 2c (-0.34) and 2i (-0.33) than for the bisaminopyrimidine
derivatives 3m (-0.29) and 3n (-0.26). Furthermore, the top-scored pose of redocked
fragments 2c and 2i resemble the orientation of sunitinib in cocrystal structures, where the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
-carbon of pyrrole is exposed to the solvent site of the ATP binding pocket (Figure S26).
Irreversible indolinone scaffolds 2c and 2i thus were merged with the parent inhibitor
sunitinib by addition of a 2- and 4-methyl group to the pyrrole ring as well as a 3-
carboxamide-linked ethylene diethylamine group to give the hybrid structures 4a and 4b
(Figure 2c). Synthesis was performed similar to procedures described before (SI chapter 2h).
Redocking in FLT3 DFG-in was performed using a 2 Å pharmacophore of the indolinone
hydrogen bond donor, acceptor and aryl feature. The top-scored binding poses of 4a and 4b
show comparable binding modes as fragments 2c and 2i and sunitinib (Figure 2d, e; S27, S28).
The conformation of the solvent-exposed diethylamine group is highly flexible (Figure S29).
β
Receptor-free energy minimization of the ligand shows a distance of the vinyl C atom of
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
.12 Å to its position in the docked pose while the energy plot as a function of the dihedral
angle of the sulfonamide is close to its minimum (Figure S30).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Comparison of the docked (green) and energy-minimized structure (blue) of (a) the
active vinyl sulfonamide 2i and (b) the inactive acrylamide 2f. (c) Structure of 2nd-
generation indolinone inhibitors. (d) Top-scored covalent docking pose of 4a and (e) 4b.
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Table 4. Inhibition of cell viability in FLT3-mutated (MV4-11), FLT3wt (THP1) and FLT3-
negative (Jurkat) cells after 72 h treatment. Inhibition of purified kinase activity is
determined at 100 µM ATP and 30 min pre-incubation of kinase and inhibitor using the ADP
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Glo assay. Half-life towards 100-fold excess of reduced glutathione and 10 µM inhibitor as
determined by HPLC. n.d. = not determined.
IC50 / nM
THP1
Relative Inhibition / %
FLT3(D835Y) FLT3(ITD)
a
Compound
MV4-11
Jurkat
t
1/2 / min
(FLT3(ITD)) (FLT3wt) (FLT3 neg.)
c = 0.1 µM
c = 1 µM
c = 0.1 µM
4
a
21 ± 1
6.0 ± 0.3
> 1 µM
643 ± 95
> 1 µM
> 1 µM
n.d.
> 1 µM
> 1 µM
n.d.
71
90
73
218
51
4
b
95
n.d.
32
100
n.d.
n.d.
86
4c
n.d.
n.d.
n.d.
n.d.
4
d
n.d.
n.d.
3
m
2.3 ± 0.1
58 ± 16
54 ± 1
172 ±24
514 ± 8
44 ± 5
47
86
82
85
95
90
95
73
99
70
91
97
58
82
3n
298 ± 10
Sunitinib
> 1 µM
> 1 µM
> 1 µM
> 1 µM
> 1 µM
> 1 µM
96
n.d.
n.d.
n.d.
Hypothemycin
Crenolanib
15 ± 3
92
12 ± 3
100
Introduction of the 4-methyl group close to Cys694 changed the position of the
sulfonamide group, slightly forcing a strong hydrogen bond donor-acceptor interaction with
Lys644. Molecular dynamics simulation of 4b in complex with the FLT3-DFG-in homology
model over 110 ps gives only small RMS deviations (0.5 Å to 1.0 Å) compared to the initial
complex concluding that the protein-ligand complex is highly stable (Figure S31). The vinyl
sulfonamide substituted sunitinib derivative 4b shows an increase in cytotoxicity towards
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MV4-11 cells compared to the irreversible fragment 2i while the reversible ethyl
sulfonamide 4d is ~100-fold less cytotoxic. Additionally, inhibition of FLT3(D835Y) is
further increased, while inhibition decreases by the ethyl sulfonamide compared to
sunitinib. Optimization of the irreversible fragment 2c to give the sunitinib-derived α-chloro
acetamide 4a yields a 2.5-fold increased cytotoxicity while the unreactive acetamide
derivative 4c is inactive in MV4-11 cells. 4a and 4b exhibit both superior cytotoxicity to
sunitinib in MV4-11 cells while they retain selectivity towards FLT3wt THP1, NOMO1,
U937 as well as FLT3-negative Jurkat cells (Figure S32). In FLT3(ITD)-positive MOLM-14
cells 4b (IC = 7.8 nM) is more cytotoxic than 4a (IC = 26.4 nM) and 3m (IC = 11.6 nM).
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Vinyl sulfonamide 4b shows superior cytotoxicity compared to the type I inhibitors
midostaurin (~2x), crenolanib (~2x) and hypothemycin (~2.5x) in MV4-11 cells. While
increased cytotoxicity in MV4-11 cells by 4b compared to 4a correlates with decreased
pFLT3 and pSTAT5 levels, 3m shows higher levels of prosurvival-signaling correlating with
decreased inhibition in FLT3 kinase assays (Figures 3a, S33).
Reactivity towards Nucleophiles. Half-life of electrophilic compounds was compared in a
HPLC-based assay using 100-fold excess of nucleophiles to investigate the properties of the
different CRGs (Figures 3b). While the acrylamide 2f and α-fluoroacetamide 2b are highly
stable (t1/2 > 24 h), the propargylamide 2g (50 min) as well as the hit-compounds 2c (384 min)
and 2i (71 min) are less stable. Half-life is decreased by the introduction of the alkaline
tertiary amine in 4a (218 min) and 4b (52 min). Consequently, pre-incubation of 4b with a
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000x excess of glutathione (100 nM inhibitor) for 30 min decreased the inhibition of
FLT3(D835Y) kinase activity from initial 95% to 68% while incubation for 24h decreases
inhibition further (37%). Increase of buffer pH leads to a decrease of half-life (28 min, pH 8)
while a decrease of pH increases half-life (130 min, pH 6.8; Figure S34). The α-N-Cbz-
protected lysine, serine and threonine amino acids show no conversion with 4b (pH 7.4).
Danio Rerio Embryo Toxicity Assay. Biocompatibility and toxicologic effects of irreversible
FLT3 inhibitors were investigated in Danio rerio embryos, that have been shown to be a
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promising model for drug toxicity screening (Figure 3c). Interestingly, in Danio rerio the
targeted cysteine (DFG-1) is replaced by an alanine residue, suggesting that off-target rather
than FLT3-dependent toxicity can be observed (Table S6, S7). Comparison of the 2-halo-
substituted acetamides shows high toxicity for the bromo- 2d and fluoro- 2b derivative (10
µM inhibitor), while the chloro- derivative 2c as well as the reversible acetamide 2a are not
lethal up to 96 h post incubation (120 hpf). The vinyl sulfonamide 2i as well as hybrid
structures 4a and 4b are non-toxic at 10 µM comparable to sunitinib, hypothemycin and
crenolanib. The bisamino-pyrimidine 3m shows increased toxicity that is comparable to
fragment 1f suggesting a more promiscuous mode of action.
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Figure 3. (a) Western Blot analysis of FLT3(Y969) phosphorylation and downstream STAT5
phosphorylation after 1 h incubation with 4b. (b) Half-life of electrophilic inhibitors towards
reduced glutathione (100x excess, pH 7.4). (c) Toxicity of selected inhibitors on Danio rerio
embryos (96 hpf, 72 h incubation, 10 µM). (d) Inhibition of FLT3(D835Y) by 4a and 4b is
insensitive to high ATP concentrations but is (e) highly dependent on ATP concentration for
reversible inhibitors.
ATP- and time-dependent inhibition. We further investigated the ATP- and time-
dependency of kinase inhibition by compounds 4a, b and 3m, n in FLT3(D835Y)
representing the most active compounds from both series. As expected, the reversible
binding FLT3 inhibitors crenolanib and sunitinib show a strong dependency on ATP
concentration, where a 10-fold ATP increase (100 µM to 1 mM) leads to an approximately
10-fold decrease of inhibition whereas crenolanib (4.2 nM to 42 nM) remains more potent
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than sunitinib (8.2 nM to 105 nM, Figure 3d, e). Since irreversible bond formation leads to
non-equilibrium-binding only the initial reversible complex, preceding covalent bond
formation, is ATP-competitive. Indeed, inhibition of FLT3(D835Y) by 4a (67 nM at low ATP,
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8 nM at high ATP) as well as 4b (11.6 nM at low ATP to 18.6 nM at high ATP) is less
vulnerable to increased ATP concentrations. To our surprise, 3m and 3n are highly
susceptible to high ATP concentrations suggesting low inactivation kinetics and thus high
competitivity to ATP (Figure S35). Additionally, we determined the influence of the pre-
incubation time of inhibitor and kinase alone where ATP is not competing with inhibitor
binding. As expected, the indolinones 4a and 4b as well as the bisaminopyrimidines 3m and
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n show increased inhibition of FLT3(D835Y) and FLT3(ITD) when pre-incubation time is
increased from 5 min to 30 min (Table S8). To determine the K and k values of the most
i
inact
potent covalent FLT3 inhibitors 4a and 4b we evaluated time-dependent IC values against
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0
FLT3(D835Y) (Figure S36). K and k
have been obtained from the plot IC (t) vs t by
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inact
50
nonlinear regression (see experimental section). We found that 4a has a lower binding
affinity (K = 10.14 nM) than 4b (K = 1.98 nM) but a higher inactivation constant (k
=
i
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inact
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0.0033 / s and 0.0017 / s ). However, determination of K /K shows that 4b is a more
inact
i
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1
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-1 -1
potent irreversible inhibitor (kinact/K = 0.84 µM s ) than 4a (k /K = 0.33 µM s ).”
i
inact
i
Trypsin-digest and MALDI-MS of covalently modified FLT3. Since compounds 4a and 4b
represent the most active compounds in this study covalent modification of Cys828 was
confirmed by tryptic in-gel digest of FLT3wt, that was considered to yield the modified
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octapeptide IC DFGLAR that could be detected by MALDI mass spectrometry. Standard
denaturation and alkylation protocol using dithiothreitol and 2-iodo acetamide allowed
detection of the single acetamide-substituted peptide with good agreement of the calculated
and found masses and the modeled and measured isotopic pattern (Figure S37 – S41).
Subsequent MS/MS allowed detection of numerous fragments of this sequence. Pre-
incubation of FLT3wt with 2i, hypothemycin, 4a and 4b allowed detection of calculated
masses with small errors confirming covalent modification of Cys828 by the key compounds
in this study.
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Kinase selectivity of 4b and activity towards resistance-conferring mutations. We chose
compound 4b as lead structure in this project as it shows the strongest inhibition of the
isolated kinase and strongest cytotoxicity and further investigated kinase selectivity as well as
activity in drug-resistant mutations. Selectivity of the most potent inhibitor 4b was
determined in a panel of 34 kinases at 100 nM inhibitor (Figure 4a). Kinases, that are
inhibited by sunitinib, were selected for testing based on available K values for sunitinib and
d
for containing the homologous CDFG motif (Table S9, Figure S42). As expected, FLT3 (95%)
and FLT3(D835Y) (99%) are potently inhibited. Surprisingly, other kinases of the PDGF
receptor family containing the conserved DFG-1 cysteine are significantly less inhibited,
including c-KIT (34%), PDGFRα (24%) and PDGFRβ (14%). This is remarkable as they all
share high sequence similarities (>70%) to FLT3 and exhibit single-digit to sub-nanomolar Kd
values for sunitinib.32 Interestingly, less related kinases (30 to 40% sequence similarity)
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containing the CDFG motif (ERK1, ERK2, GSK3α, GSK3β) are not inhibited at 100 nM 4b
while being reported targets of hypothemycin.10 4b may also covalently modify the
homologous serine residue (DFG-1) as the Ret kinase (96%) is strongly inhibited.
Interestingly, 5-amino indolinones have been reported to be potent Ret and FLT3 inhibitors
while superposition of the FLT3 model and Ret cocrystal structure is in good accordance
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(
RMSD = 0.7 Å, Figure S43). The homolog alanine and glycine residues are tolerated by 4b
(Lyn, Lck, Axl, IRAK1; 93% to 81% inhibition) suggesting that hydrogen bond interactions
to the conserved lysine may account for increased affinity compared to sunitinib while a
γ
γ
steric clash with CysS or SerO is prevented. We further assessed our lead compound 4b
using the KinomeScan screening platform, where numerous FLT3 inhibitors have been
assessed before (Figure 4b).34-35
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Figure 4. (a) Selectivity of 4b in a panel of 34 kinases. (b) K values of 4b and inhibitors in
d
clinical trials in a panel of FLT3-mutants. (c) Relative inhibition by time (IC (crenolanib)/
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IC (4b)). (d) Effect of inhibitor wash-out after 2 h, 8 h, 24 h incubation and read-out at 72 h
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Sunitinib and crenolanib show higher affinity towards FLT3wt and FLT3(ITD) than 4b,
while midostaurin is less efficient. Interestingly, sunitinib showed a ~6-fold increased K in
d
FLT3(ITD) but inhibition despite cell viability in FLT3-ITD was ~10-fold increased for 4b.
Crenolanib and quizartinib lose affinity towards dual ITD and point-mutated FLT3, while
affinity is increased for 4b. Especially towards the drug-resistant FLT3(ITD, F691L) mutation,
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b shows a ~15-fold increased affinity compared to crenolanib. Comparison of the affinity of
4b for the non-autoinhibited (K = 2.3 nM) and the autoinhibited state (K = 65 nM) of FLT3
d
d
shows a 28-fold decrease that is similar to type I inhibitors as sunitinib and midostaurin
while type II inhibitors like sorafenib and quizartinib typically give a 100- to 1000-fold shift
in affinity.37
Time-dependent inhibition of cell viability. To monitor the onset of cytotoxicity we
compared crenolanib and 4b in a real-time cell viability assay with MV4-11 cells (Figure 4c,
Figure S44). While cytotoxicity became detectable already after 2 h, robust fitting of cell
viability IC50 was possible after 8 h for both compounds. Cytotoxicity by 4b relative to
crenolanib was maximal at 18 h (4.4-fold) and plateaus to ~1.5x stronger inhibition for 4b
after 24 h until the end of the assay (77 h). Interestingly, short incubation times (2 h, 8 h)
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followed by wash-out of the inhibitor and incubation in inhibitor-free medium negatively
affects the performance of 4b compared to crenolanib (Figure 4d). However, potency of 4b is
increased by incubation times longer than 24 h suggesting that rapid de novo synthesis of
FLT3-ITD may lead to resistance to short incubation times of irreversible inhibitors.
However, onset of inhibition by 4b demonstrated by decreased pFLT3 and downstream
pSTAT5 levels is in the single-digit nanomolar range after 1 h (Figure 3a).
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Evaluation of BODIPY-labeled Activity-based Probes. We further aimed to modify the hit
compounds 2c and 2i with a boron-dipyrromethene (BODIPY) label attached to the solvent-
exposed site of the fragment to image irreversible protein binding similar to a recently
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reported Ibrutinib-based probe (Figure 5a, b). Synthesis was performed similar to reported
work.
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