Discovery and Optimization of wt-RET/KDR-Selective Inhibitors of RETV804M Kinase

Article abstract of DOI:10.1021/acsmedchemlett.9b00615

A combination of focused library and virtual screening, hit expansion, and rational design has resulted in the development of a series of inhibitors of RET^V804M kinase, the anticipated drug-resistant mutant of RET kinase. These agents do not inhibit the wild type (wt) isoforms of RET or KDR and therefore offer a potential adjunct to RET inhibitors currently undergoing clinical evaluation.

Full text of DOI:10.1021/acsmedchemlett.9b00615

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Letter
Discovery and Optimisation of wt-RET/
V804M
KDR-selective Inhibitors of RET Kinase
Rebecca Newton, Bohdan Waszkowycz, Chitra Seewooruthun, Daniel Burschowsky, Mark Richards,
Samantha Hitchin, Habiba Begum, Amanda Watson, Eleanor French, Niall Hamilton, Stuart Jones,
Li-Ying Lin, Ian Waddell, Aude Echalier, Richard Bayliss, Allan M. Jordan, and Donald Ogilvie
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/
acsmedchemlett.9b00615 • Publication Date (Web): 28 Feb 2020
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ACS Medicinal Chemistry Letters
Discovery and Optimisation of wt-RET/KDR-selective Inhibitors of RETV804M Kinase
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Rebecca Newton,1,* Bohdan Waszkowycz, Chitra Seewooruthun, Daniel Burschowsky, Mark Richards, Samantha
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Hitchin, Habiba Begum, Amanda Watson , Eleanor French, Niall Hamilton, Stuart Jones, Li-Ying Lin, Ian
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Waddell, Aude Echalier, Richard Bayliss, Allan M. Jordan and Donald Ogilvie.
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1Drug Discovery Unit, Cancer Research UK Manchester Institute University of Manchester, Alderley Park,
Macclesfield, SK10 4TG. U.K.
2Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester, Lancaster Road,
Leicester, LE1 7RH, UK.
3Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2
9
JT, UK.
4Leicester Drug Discovery & Diagnostics Centre (LD3), R407a, Hodgkin Building, Lancaster Road, Leicester, LE1
HB, UK.
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ABSTRACT
A combination of focussed library and virtual screening, hit expansion and rational design has resulted in the
development of a series of inhibitors of RETV804M kinase, the anticipated drug-resistant mutant of RET kinase. These
agents do not inhibit the wild type (wt) isoforms of RET or KDR and therefore offer a potential adjunct to RET
inhibitors currently undergoing clinical evaluation.
Keywords: RET, KDR, kinase inhibitor, virtual screening
Activating gene fusions of the receptor tyrosine kinase RET have recently been identified as drivers in around 1-2% of
all non-small cell lung cancers (NSCLC).1-3 In non-smokers, the incidence of these mutations rises to around 6%.4
Following the clinical success of similar agents targeting other lung cancer driver mutations, such as EGFR and ALK,
there has been a recent resurgence of interest in developing inhibitors of the RET kinase domain.
Whilst the clinically approved agents vandetanib 1 and cabozantinib 2 are being extensively evaluated in clinical trials
for this indication, RET inhibition is a secondary pharmacology of these compounds. The primary pharmacology of
these agents, namely inhibition of KDR (or VEGFR2), leads to dose-limiting side effects, significantly compromising
their therapeutic utility in NSCLC.5,6 Given this observed clinical toxicity, more selective RET inhibitors, devoid of
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KDR inhibitory activity in vivo, have been of considerable interest and agents such as LOXO-292 3 and BLU-667 4
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are now being evaluated in clinical trials (Figure 1).7,8
N
Br
N
N
NH
N
O
F
OMe
O
OH
N
MeO
N
N
N
N
NMe
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H
N
N
O
N
N
O
O
N
F
O
N
H
OMe
OMe
OMe
N
N
NH
F
HN
N
N
2
4
Figure 1: Exemplar first generation non-selective and second generation (KDR-selective) RET inhibitors.
However, patients treated with kinase inhibitors are frequently prone to early clinical relapse due to mutations in the
kinase ATP binding domain, which render the protein catalytically active but no longer sensitive to drug treatment.
Such mutations often occur in the so-called “gatekeeper” region. In the case of RET, resistance is predicted to arise
9
from a Val to Met or Leu mutation at residue 804. Anticipating this outcome, second generation RET-selective
inhibitors 3 and 4 also inhibit the gatekeeper mutant (RETV804M/L) kinase domain.7,8
At the outset of this study, clinical data for selective RET inhibitors was unavailable, and as such the dose-limiting
toxicity of a truly RET-selective inhibitor was unknown. Drawing on the precedent of inhibitors of EGFR in lung
cancer, where wild type-sparing mtEGFR inhibitors offer significant clinical advantage,10 we hypothesised that a
RETV804M/L-selective, rather than mixed wild type/gatekeeper mutant inhibitor, may offer an alternate therapeutic
option for patients with refractory disease, particularly if robust RET inhibition was shown to induce significant
tolerability issues. Such an agent would allow more robust inhibition of the target and may deliver enhanced
therapeutic benefit. Further, a RETV804M selective agent may serve as a useful adjunct therapy alongside RET-selective
agents, which do not maintain activity against the anticipated gatekeeper resistance mutants.11
To deliver RETV804M-selective inhibitors, we decided to implement screening of a commercial kinase-directed library
of just over nine thousand compounds, alongside a virtual screening cascade based on docking to in-house RETV804M
homology models, derived in turn from published wild type RET kinase domain crystal structures (PDB 2IVU and
2
IVT). We reasoned that the mutated gatekeeper sidechain, replacing a small Val by a larger Met/Leu, occludes access
to the hydrophobic pocket normally exploited by compounds such as 1, hence their loss of binding affinity for
V804M 12
RET
. We therefore sought to identify ligands that avoided this pocket and bound primarily to the site otherwise
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ACS Medicinal Chemistry Letters
occupied by ATP. Whilst such ligands retain the potential to inhibit RET, our intention was primarily to identify
ligands that would not be penalised by the mutation, though we recognised that there was a risk of reducing selectivity
over other kinases, as the hydrophobic pocket contributes to the kinome selectivity profile of compounds such as 1.
However, we postulated that the desired selectivity over RET might then arise by virtue of enhanced contacts between
the ligand and the more hydrophobic mutated gatekeeper, or (more serendipitously) by a particular scaffold potentially
exploiting other more subtle structural differences between the two binding sites.
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To this end, a library of 984,000 lead-like compounds (internally triaged from the eMolecules screening collection )
was docked against our homology models (Glide HTVS and SP docking, Schrödinger, LLC, New York) and manual
triage of the resultant hits undertaken, with selection based upon factors including docking score, maintenance of the
key hinge-binding interactions, clustering by 2D chemotype and visual assessment of binding poses, assessing both
novelty of interactions and potential for selectivity against RET and KDR. From this exercise, we selected a set of 197
compounds, for purchase and screening in biochemical assays against our three key targets. Pleasingly, several of
these early derivatives were found to show promising selectivity of up to 100-fold against both RET and KDR (Figure
2
a) and these clustered predominantly into five distinct chemotypes. These encouraging start-points (four clusters
derived from virtual screening and one from the kinase-focussed library) were further elaborated through both
analogue-by-catalogue hit expansion and modelling-directed analogue synthesis, rapidly delivering both increased
potency and selectivity against key off-targets across several series (Figure 2b).
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Figure 2: a) Selectivity profile of hits obtained from initial kinase library and virtual screen; b) Hit expansion through
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analogue by catalogue and focussed synthesis. In both cases, plots display RETV804M biochemical pIC vs wt-RET
5
0
pIC50 (upper panel) and KDR pIC50 (lower panel). Colours indicate the five identified chemotypes of interest.
Desirable hits are found in the lower right quadrant of each plot.
From these early studies, we became particularly interested in hits derived from a pyrazolopyrimidine scaffold (Figure
0
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, orange dots), exemplified by the benzamide analogue 5 (Figure 3). Whilst related pyrazolo[3,4-d]pyrimidin-4-
1
4
15,16
amines have been described previously by Yoon and others,
these derivatives have been generally shown to be
more potent inhibitors of wild type RET than RETV804M. This observation perhaps makes the initial selectivity profile
observed with this early pyrazolo[1,5-a]pyrimidin-5-amine all the more surprising. Docking of compound 5 (Figure
4a) suggested an attractive binding mode with options to enhance both RET and KDR selectivity. We predicted that
the pyrazolopyrimidine core would bind to the hinge region, directing the benzamide towards the catalytic lysine
(Lys758) and placing the pyridine in the pocket normally occupied by the ribose moiety of ATP. Both of these sites
offer opportunities for optimising protein-ligand interactions to improve affinity and selectivity; for example, the
ribose pocket is bounded by residues Ser811 and Ser891 which are not conserved between RET and KDR. Our
interest was further enhanced after biochemical evaluation, which demonstrated the compound to be over 100-fold
selective against KDR and almost 4-fold selective against RET. The compound also demonstrated modest cellular
potency against our RETV804M cell assay, albeit with a considerable decrease in selectivity.
O
NH2
N
H
N
N
N
N
5
Figure 3: Early pyrazolopyrimidine hit 5.
To permit exploration of structure-activity relationships, analogues of 5 were prepared from commercially available
pyrazolopyrimidine 6, through S Ar displacement to give intermediates 7-10 and Pd-mediated coupling, as described
N
in Scheme 1. 1 and 2 were used as benchmark compounds for our subsequent studies. As shown in Table 1, both were
more active against wild type RET and KDR than they were against RETV804M. Early structure-activity relationship
investigations suggested that the amide motif of 5 was important for binding, with the dimethyl derivative 11 showing
a significant loss of activity in our biochemical assay (Table 1). The regiochemistry of the pendant pyridyl moiety was
also investigated (compounds 12 and 13), though the position of the nitrogen did not result in any significant alteration
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ACS Medicinal Chemistry Letters
to potency or selectivity, suggesting that the nitrogen atom itself does not participate in a specific, directional
hydrogen-bonding interaction. However, the simple incorporation of a basic nitrogen in a similar spatial region
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6
(compound 14) resulted in a ten-fold reduction in potency.
Scheme 1: Synthesis of compounds 5, 11-23.
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R2
Br
Br
H
H
Cl
N
i)
N
N
ii)
N
N
R1
R1
N
N
N
N
N
N
6
7-10
5, 11-23
Reagents and conditions: i) R -NH , DIPEA, IPA, 100 °C, 5h, 47-100%; ii) arylboronic acid, K PO , Pd-118, 1,4-
1
2
3
4
dioxane, water, µwave, 120 °C, 30 min, 7-34%
Further studies confirmed that removal of the amide functionality (compound 15) resulted in a significant loss of
biochemical potency as did movement of the amide from the para- to meta-position (compound 16) (Table 2). The
corresponding sulphonamide 17 and carboxylic acid 18 were reasonably well tolerated. Reversal of the amide or
sulphonamide (compounds 19-21) was detrimental to potency. These results were generally consistent with our
hypothesised binding mode, suggesting that specific H-bonding interactions between the benzamide of 5 and Lys758
were important for potency. Further exploration of the SAR around this ring gave disappointing results (data not
shown), but led to the suggestion to mimic the primary amide in a bicyclic system. These investigations led to 22 and,
whilst this compound was ten-fold less potent than 5 in biochemical assays, RET selectivity was improved
considerably. Of more interest, 5 and 22 were roughly equipotent in our RETV804M cell assay, despite the differences in
biochemical activity. Whilst this cellular selectivity was only a modest two-fold at this stage, we felt that this
derivative offered potential for further gains and a wider exploration of bicyclic systems was undertaken, leading to
2
3. Whilst this compound appeared less impressive in terms of selectivity in our biochemical assays than 22, it did
display moderately improved cell potency and slightly improved cell selectivity against both the RET and KDR driven
cell lines.
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Table 1: Initial optimisation of hit pyrazolopyrimidine 5.a
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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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
O
R2
H
N
N
R1
N
N
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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5
6
7
8
9
0
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9
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5
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9
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Biochemical Biochemical RETV804M Cell assay Cell assay
RETV804M
selectivity,
selectivity,
cellular
IC50
selectivity, selectivity,
Compound
R1
R2
biochemical
IC50 (µM)
RETV804M vs RETV804M vs
RETV804M
vs RET
0.24
RETV804M
vs KDR
0.43
RET
KDR
(µM)
1
2
-
-
-
-
>5
0.009
0.03
1.5 (0.92)
0
.18
1.5 (2.4)
0.33
0.01
1.1
0.08
2.3
(0.069)
5
NH
2
0.02 (0.007)
3.7
1.8
110
3.9
4.4 (0.18)
0.89
N
N
11
NMe
2
7.6 (1.0)
0.044
N
1
2
NH
2
19
22
3.7 (0.41)
0.90
2.0
(0.014)
0.051
3.9
N
1
1
3
4
NH
NH
2
2
13
19
54
0.85
0.30
1.9
(
0.0078)
(0.20)
N
0.35 (0.034)
4.7
> 10
0.83
aAll assay values are the geometric mean of at least two independent duplicate determinations, quoted to two significant figures.
Standard deviations are given in parentheses. Biochemical and cellular selectivities are expressed as fold-values.
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Table 2: Further optimisation of hit pyrazolopyrimidine 5.a
1
2
3
4
5
6
7
8
9
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
N
R
H
N
N
N
N
Biochemical Biochemical RETV804M Cell assay Cell assay
RETV804M
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
selectivity,
selectivity,
cellular
IC50
selectivity, selectivity,
Compound
R
biochemical
IC50 (µM)
RETV804M vs
RETV804M vs
RETV804M
RETV804M
RET
KDR
(µM)
vs RET
vs KDR
O
0
.025
5
NH2
3.7
110
4.4 (0.18)
0.89
2.3
(0.007)
1
1
5
6
Ph
1.14 (0.43)
4.3 (1.05)
2.4
0.96
8.0
NH2
n/d
O
O
S
O
1
1
7
8
NH2
OH
0.36 (0.14)
0.56 (0.19)
1.3
1.2
220
1.8
> 10
1
1
O
H
N
1
2
9
0
6.7 (1.72)
1.6 (0.15)
1.5 (0.89)
2.6
4.0
4.4
22
29
O
O
N
H
H
N
O
S
21
0.28
O
N
2
2
0.30 (0.065)
0.29 (0.16)
18
83
27
2.8 (0.19)
1.5 (0.03)
1.9
2.2
1.9
6.2
N
H
O
O
23
0.64
aAll assay values are the geometric mean of at least two independent duplicate determinations, quoted to two significant figures.
Standard deviations are given in parentheses. Biochemical and cellular selectivities are expressed as fold-values.
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Similar to many of the derivatives prepared to this point, 23 suffered from modest kinetic solubility (30 µM) and
further optimisation was undertaken in an effort to optimise both the cellular activity profile and solubility in parallel.
Methods used to synthesise these derivatives are described in Scheme 2. Briefly, formylation of peperacetonitrile with
ethyl formate and cyclisation with semicarbazide gave the aminopyrazole 24, which was further cyclised to 25 and
1
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9
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
halogenated to give 26. Finally, S Ar displacement gave the desired target compounds.
N
0
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9
0
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5
6
7
8
9
0
Scheme 2: Synthesis of compounds 27-35.
O
O
O
O
O
O
O
O
O
O
i)
ii)
H
iii)
iv)
H
H N
2
O
N
Cl
N
N
N
N
R
HN
N
N
N
N
N
N
N
24
25
26
27-35
Reagents and conditions: i) a) Ethyl formate, NaOMe, PhMe, reflux, 24h; b) semicarbazide, methanol, 0 °C, 1h; c)
NaOH, 100 °C, 30 min, 55% overall; ii) 1,3-dimethyluracil, NaOEt, EtOH, reflux,24h, 83%; iii) POCl , DMF, reflux,
3
1
.5h, 78%; iv) amine, DMF, 80 °C, 1h, 11-86%.
In this series of derivatives, alteration of the pyridine regiochemistry improved biochemical potency, but had little
effect on cellular activity (compound 27, Table 3). The importance of both the basic nitrogen and its positioning, were
highlighted through the significant loss of potency observed with 28 and 29 respectively, in contrast to the flat SAR
observed with 12 and 13. Saturation of the ring system (compounds 30 and 31) maintained biochemical potency.
Further investigation of these saturated systems led to the open chain amine derivative 34, which showed a greater
than ten-fold increase in cell activity compared to 23. More importantly, this compound was significantly more
selective when assessed in our RET and KDR cell assays, exceeding 25-fold selectivity against both counter-targets
for the first time. Whilst more extensive biochemical kinase profiling has not been undertaken on this derivative to
fully understand its selectivity profile, the lack of off-target cellular cytotoxicity (4 µM in RET and 9 µM in KDR vs
1
00 nM in RETV804M cell assays) suggests that even this relatively unoptimised derivative is not a promiscuous
inhibitor. Compound 34 also showed promising physicochemical properties (kinetic solubility >100 µM; Papp 22.8 ×
-
6
1
6
0 cm/s and efflux ratio 1.4 in a Caco-2 assay) but poor metabolic stability in mouse liver microsomes (MLM CL =
int
2 µL/min/mg).
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Table 3: Further optimisation of methylenedioxypyrazolopyrimidine compound 23.a
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2
3
4
5
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7
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9
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
O
O
H
N
N
R
N
N
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
Biochemical
selectivity,
RETV804M vs
RET
Biochemical
selectivity,
RETV804M vs
KDR
Cell assay Cell assay
selectivity, selectivity,
RETV804M
RETV804M
cellular
Compound
R
biochemical
IC50 (µM)
RETV804M
vs RET
RETV804M
vs KDR
IC50 (µM)
2
2
3
7
0.29 (0.16)
0.039 (0.03)
29 (14.6)
0.64
14
27
100
2.3
2.1
1.5 (0.03)
1.1 (0.03)
2.2
3.1
6.2
7.6
N
N
28
0.16
2.1
N
2
3
3
9
0
1
63 (4.2)
1.3
0.20 (0.019)
0.88 (0.15)
1.2
1.2
21
64
17
0.85
2.6
HN
(0.021)
HN
0
.74
32
N
0.091 (0.091)
0.66 (0.070)
540
34
9.8
13
(0.049)
3
3
3
4
5.3
N
0
.11
N
0.019 (0.004)
0.13 (0.012)
16
15
410
19
32
76
(0.045)
HO
35
6.8 (0.13)
1.5
1.5
aAll assay values are the geometric mean of at least two independent duplicate determinations, quoted to two significant figures.
Standard deviations are given in parentheses. Biochemical and cellular selectivities are expressed as fold-values.
Further confirming target engagement, compounds 13 and 34 were soaked into crystals of the kinase domain of
RETV804M (Figure 4b, c). Pleasingly, the crystal structure for 13 accurately recapitulated the binding mode and key
interactions predicted by docking, with the benzamide hydrogen-bonding to Lys758, Glu775, the Asp892 backbone
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and the pyridine occupying the ribose pocket. However, we were surprised to observe that 34 exhibited a different
binding mode, with the pyrazolopyrimidine core adopting a flipped orientation with respect to the hinge, while
maintaining a single hinge H-bond to Ala807 backbone amide. The tertiary amine of 34 H-bonds to Asp892 and forms
lipophilic contacts with Phe735 and Leu881, while the piperonyl moiety binds somewhat loosely between the walls of
the ATP-binding site, bounded by Leu730/Gly731 of the P-loop and Gly810/Ser811 of the ribose pocket, but without
making any significant polar interactions. We speculate that the flipping of the binding mode may be a consequence of
the binding interactions achieved by the peripheral substituents rather than the bicyclic hinge binder itself. Hence the
main driver for the binding orientation of 13 may be the hydrogen-bonding interactions between the benzamide and
Lys758/Glu775; these favourable interactions cannot be replicated by the piperonyl moiety of 34, and thus the flipped
binding mode allows for favourable polar and lipophilic interactions from the dimethylamino propyl moiety to
Asp892 and neighbouring residues. [We note that both binding orientations for a chemically similar hinge binder can
be observed in other kinase X-ray structures on the PDB: for example, 4Z7H, 4YMJ and 3BQR adopt a similar
orientation to 13, while 4TXC and 6AYW bind similarly to 34.] This alternate binding mode likely also accounts for
the differing SAR trends around the pyridylmethyl moiety in the piperonyl series (compounds 23, 27), compared to
the corresponding benzamide derivatives compounds 5, 12).
1
2
3
4
5
6
7
8
9
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
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5
5
5
5
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5
5
5
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0
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9
0
It is difficult to rationalize fully the improved selectivity of 34 over wild type RET, as there may be many contributing
factors arising from the change in substituents and the change in binding mode which would need to be explored by
more extensive SAR. The flipped orientation of the hinge binder may result in improved lipophilic contacts with the
Met gatekeeper or allow the gatekeeper to adopt a more favoured conformation. The lipophilic amine of 34 is situated
in fairly close contact to Ser891 (Figure 4d), and this may contribute to the improved selectivity over KDR (which
features a larger Cys sidechain in the equivalent position). Whatever the rationale, it is of interest that the flipped
binding orientation has led to diverging SAR for this series.
To our knowledge, this is the first report of RET- and KDR-selective inhibitors of RETV804M, assessed through both
biochemical and cellular assays. We believe these compounds may offer useful starting points in the delivery of novel
adjunct therapeutics, delivering additional patient benefit alongside the RET inhibitors presently in clinical
development.
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a)
b)
c)
d)
Figure 4: a) Docked model of early hit 5 bound to RETV804M; b) Crystal structure of 13 bound to RETV804M (PDB
I83); c) Crystal structure of optimised lead 34 bound to RETV804M (PDB 6I82); d) 2D Ligand interaction plot for 34
6
bound to RETV804M. In all panels, RETV804M is shown as grey cartoons, key residues as yellow sticks and the respective
ligand as green sticks.
ASSOCIATED CONTENT.
Supporting Information Assay protocols, purity data for all compounds, general experimental details and
representative synthetic procedures. This material is available free of charge at http://pubs.acs.org
Accession Codes
RETV804M kinase domain in complex with 13, 6I83; RETV804M kinase domain in complex with 34, 6I82.
Author information
*Telephone: +44 (0)161 3066272. Email: rebecca.newton@cruk.manchester.ac.uk.
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Author contributions.
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R.N., B.W. and A.J. wrote the manuscript. R.N., B.W., E.F., N.H., S.J. and A.J. designed and/or synthesised
compounds. B.W. conducted the computational aspects of the study. C.S., D.B., M.R., L-Y.L., A.E. and R.B.
conducted protein production, crystallography and structure refinement. S.H., H.B., A.W., I.W. and D.O. designed and
conducted the biological screening cascade.
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Notes
The authors declare no competing financial interests.
ACKNOWLEDGEMENT
This work was supported by Cancer Research UK (Grant C480/A11411 and C5759/A17098) and the CRT Pioneer
Fund, managed by Sixth Element Capital LLP. We thank the CRUK Centre Network Structural Biology Accelerator
Award (Grant number C1362/A20263) for access to structural biology capabilities and expertise. JChem for Excel
was used for structure property prediction and calculation and general data handling (JChem for Excel, version
6
.1.5.781, 2008−2013, ChemAxon (http://www.chemaxon.com). Images of protein-ligand docking were captured
within the PyMOL Molecular Graphics System, Version 2.3.4 (Schrödinger, LLC, New York) and the 2D ligand-
protein interaction diagram was prepared using Maestro (Schrödinger, LLC, New York). We thank Diamond Light
Source for access to beamlines i04 & i04-1 (MX-8997) which contributed to the results presented here. HRMS data
were generated by Gareth Smith at the University of Manchester.
ABBREVIATIONS
ALK, anaplastic lymphoma kinase; ATP, adenosine triphosphate; EGFR, epithelial growth factor receptor; HTVS,
high-throughput virtual screening; KDR, kinase insert domain; NSCLC, non-small cell lung cancer; RET, re-arranged
during transfection; SAR, structure-activity relationship; SP, standard precision virtual screening.
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Discovery and Optimisation of wt-RET/KDR-selective Inhibitors of RETV804M Kinase
Rebecca Newton,1,* Bohdan Waszkowycz, Chitra Seewooruthun, Daniel Burschowsky, Mark Richards, Samantha
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Waddell, Aude Echalier, Richard Bayliss, Allan M. Jordan and Donald Ogilvie.
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