Fragment-Based Discovery of Novel Allosteric MEK1 Binders

Article abstract of DOI:10.1021/acsmedchemlett.0c00563

The MEK1 kinase plays a critical role in key cellular processes, and as such, its dysfunction is strongly linked to several human diseases, particularly cancer. MEK1 has consequently received considerable attention as a drug target, and a significant number of small-molecule inhibitors of this kinase have been reported. The majority of these inhibitors target an allosteric pocket proximal to the ATP binding site which has proven to be highly druggable, with four allosteric MEK1 inhibitors approved to date. Despite the significant attention that the MEK1 allosteric site has received, chemotypes which have been shown structurally to bind to this site are limited. With the aim of discovering novel allosteric MEK1 inhibitors using a fragment-based approach, we report here a screening method which resulted in the discovery of multiple allosteric MEK1 binders, one series of which was optimized to sub-μM affinity for MEK1 with promising physicochemical and ADMET properties.

Full text of DOI:10.1021/acsmedchemlett.0c00563

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Fragment-Based Discovery of Novel Allosteric MEK1 Binders
Paolo Di Fruscia,* Fredrik Edfeldt,* Igor Shamovsky, Gavin W. Collie, Anna Aagaard, Louise Barlind,
̈
Ulf Bo
̈
rjesson, Eva L. Hansson, Richard J. Lewis, Magnus K. Nilsson, Linda Oster, Josefine Pemberton,
k*
Lena Ripa, R. Ian Storer, and Helena Kac
̈
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sı Supporting Information
ABSTRACT: The MEK1 kinase plays a critical role in key cellular processes, and as such, its dysfunction is strongly linked to
several human diseases, particularly cancer. MEK1 has consequently received considerable attention as a drug target, and a
significant number of small-molecule inhibitors of this kinase have been reported. The majority of these inhibitors target an allosteric
pocket proximal to the ATP binding site which has proven to be highly druggable, with four allosteric MEK1 inhibitors approved to
date. Despite the significant attention that the MEK1 allosteric site has received, chemotypes which have been shown structurally to
bind to this site are limited. With the aim of discovering novel allosteric MEK1 inhibitors using a fragment-based approach, we
report here a screening method which resulted in the discovery of multiple allosteric MEK1 binders, one series of which was
optimized to sub-μM affinity for MEK1 with promising physicochemical and ADMET properties.
KEYWORDS: MEK1, Kinases, Allosteric, COPD, Fragments, FBDD
he MAP kinase signaling pathway influences many key
cellular processes including proliferation and differ-
T
entiation, and as such, dysregulation of this pathway is
known to play a major role in several human diseases,
2
,3
particularly cancer. Consequently, multiple components of
this pathway have been studied intensely as drug targets. In
particular, several inhibitors of the MEK1 kinase have now
been approved by the FDA and EMA for the treatment of
various types of cancer, with additional molecules in clinical
4
−7
and preclinical studies.
Interestingly, the MEK inhibitors
approved for clinical use are based on a shared 2,4-
dihalogenated anilido motif (Figure 1) and are known or
expected to bind to an allosteric pocket adjacent to the ATP
binding site of the kinase. The targeting of an allosteric site of a
kinase with a small-molecule inhibitor is particularly
interesting, as this potentially circumvents the expected
selectivity challenges associated with targeting the highly
conserved orthosteric ATP binding site.
Figure 1. Structures of selected approved allosteric MEK1 inhibitors.
Received: October 20, 2020
Accepted: January 25, 2021
Published: January 27, 2021
While the major focus of MEK1 as a therapeutic target has
been for oncology indications, there is additional interest in the
inhibition of this enzyme for the treatment of chronic
©
2021 American Chemical Society
https://dx.doi.org/10.1021/acsmedchemlett.0c00563
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Table 1. SPR Analysis of Fragment Hits Identified from
NMR-Based Screening of MEK1
a
Kd values are the geometric mean ± SEM of two or more
b
determinations. LE (kcal/(mol HA)): 1.37pK /HA (heavy atom
count). LipE: pK − log D. SPR pK in the presence of AMP-PNP
used in efficiency metrics. log D measured via shake-flask method in
d
c
d
d
octanol and water at pH 7.4.
obstructive pulmonary disease (COPD). −10 While approved
MEK1 inhibitors have proven to be highly effective in
oncology settings, the differing requirements of COPD small-
molecule therapeuticsfor example, the desire for rapid
systemic clearance of an inhaled drugsuggest that new
MEK1 inhibitors with differing physicochemical and ADME
properties may be needed, while retaining the benefits of an
allosteric binding mode. Interestingly, fragment-based meth-
8
1
1
ods have been employed as a means to discover allosteric
1
2−17
18,19
inhibitors of various protein classes,
including kinases,
Figure 2. Crystal structures of MEK1 bound by compounds 4 (a) and
6 (b). (c) Comparison of binding modes of 6 (blue carbons) and
cobimetinib (PDB entry 4AN2 ) (white carbons) for MEK1.
Hydrogen bonds to MEK1 are shown as blue or white dashes for 6
and cobimetinib, respectively. AMP-PNP: adenylyl imidodiphosphate.
AMP-PCP: adenylyl methylenediphosphonate.
yet reports of fragment screens targeting MEK1, while
successful, have yielded binders of the orthosteric ATP binding
1
2
0,21
site only.
As a consequence, we set out to design and
execute a fragment-based screening campaign targeting MEK1
specifically designed to find novel, allosteric inhibitors. These
efforts led to the discovery of several novel allosteric MEK1
binders, one series of which was optimized to sub-μM affinity
with promising physicochemical and ADMET properties
aligned with an inhaled small-molecule therapeutic.
142 fragments (14%) were identified as potential allosteric
binders.
In an effort to find novel allosteric MEK1 binders by
fragment-based methods, we first selected a fragment library
tailored to the MEK1 allosteric site using virtual screening
The binding affinity of these fragments for human full-length
MEK1 was then assessed by surface plasmon resonance (SPR)
in both the absence and presence of AMP-PNP. 46
2
4
22
methods. From a library of around 15k fragments, 1k were
Fragments (5%) displayed similar affinities under both
selected based on their complementarity (assessed by ligand−
conditions and yielded K values in the presence of AMP-
d
23
receptor docking studies) to the allosteric site of MEK1.
A
PNP of ≤1 mM. These were considered to be likely
noncompetitive allosteric MEK1 binders. Only two fragments
showed a significant preference for MEK1 in the absence of
AMP-PNP (i.e., AMP-PNP competition), indicating that the
screening strategy was successful in selecting noncompetitive
binders. Given the structural similarity of reported MEK1
inhibitors, the hit rate was surprisingly high yet confirms that
this is a highly ligandable binding site. Details of the most
interesting hits, based on affinity data, structural novelty, and
physicochemical properties, can be found in Table 1.
multistep 1D NMR protocol was then used to screen these
compounds for binding to MEK1. Fragments were screened in
cocktails of 6 compounds against MEK1, with 1 mM AMP-
PNP (a nonhydrolyzable analogue of ATP) included as a
means to occupy the orthosteric ATP binding site and thereby
bias the detection toward allosteric binders. Fragments
identified as positive binders under these conditions were
then subject to a competition experiment using the known
6
MEK1 allosteric inhibitor binimetinib. Using this method,
3
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Figure 3. Preliminary optimization of MEK1 primary fragment hits. (a) Compound 9 was discovered following analogue searching of our internal
chemical library using 5 as a template. A crystal structure was determined for 9 bound to MEK1, confirming an allosteric binding mode (top right
panel). (b) Compound 10 was discovered by similar methods as 9 and was used as a template for a library synthesis aimed at further improving
affinity for MEK1 (see Scheme 1 and Table 2 for further details).
a
Scheme 1. General Synthetic Route of Compounds 11−22
alternative scaffolds. Consequently, employing the NNS
25
technology, we searched our internal chemical library for
close analogues of all of the primary hits, with selected
compounds assessed by SPR to measure affinities for MEK1.
These efforts yielded two stand-out binderscompound 9,
with an affinity of 28 μM, and compound 10, with an affinity of
6
5 μM (Figure 3).
Crystallization attempts with MEK1 were attempted for
both compounds, which yielded a crystal structure of
compound 9 (Figure 3a, right panel). This structure shows
compound 9 binding to the allosteric pocket, with the
interaction seemingly primarily driven by hydrophobic
contacts, with polar contacts to the protein limited to indirect
interactions mediated via water bridges. Although we were
unsuccessful in obtaining structural data for 10, the novelty of
this compound (particularly the oxothiomorpholine group),
coupled to the synthetic tractability of the scaffold, led us to
synthesize a chemical library around the central amide
functionality, in an attempt to improve binding affinity for
MEK1 (Figure 3b and Scheme 1).
Final library compounds were prepared in high overall yield
from commercially available 2-bromosuccinic acid (I). The
synthesis commenced by converting building block I into its
corresponding methyl diester II by treatment with iodo-
methane and potassium carbonate in DMF in moderate yield
a
Reagents and conditions: (a) MeI, K CO , DMF, rt, 3 h. (b)
2
3
Cysteamine, MeOH, rt, 2 h. (c) K CO , MeOH, 60 °C, 5 h. (d)
NaOH, H O, MeOH, rt, 3 h. (e) Aromatic amine, HATU, DIPEA,
DMF, rt, 3 h.
2
3
2
We next turned to X-ray crystallography in order to assess
the binding mode of the identified AMP-PNP-noncompetitive
binders. Crystallization attempts (in the presence of AMP-
PNP) of all hits yielded crystal structures of compounds 4 and
6
bound to MEK1, both of which revealed the fragments to
bind to the allosteric site as anticipated (Figure 2a,b). Both
compounds localize to the allosteric pocket that sits between
the ATP binding site and the αC-helix, with both forming
strong hydrogen bonds to the backbone NH of residue S212 of
the activation loop (A-loop). Compound 6 forms an additional
hydrogen bond to the backbone carbonyl of F209, which may
explain the improved affinity and ligand efficiency of this hit
compared to 4 (Table 1). Comparison of the binding mode of
(
53%). The 3-oxothiomorpholine core was successfully
constructed with a one-pot, two-step procedure in good yield
59%): reaction with cysteamine in MeOH furnished
(
precursor III, and ring closure was subsequently accomplished
by adding potassium carbonate and heating the resultant
solution at 60 °C for 5 h. Saponification of methyl ester IV
with aqueous NaOH in MeOH afforded carboxylic acid V,
which was then converted, with appropriate amines, into the
corresponding desired final compounds employing HATU as
the peptide coupling agent in DMF (Scheme 1). Final
compounds were then assessed by SPR to measure binding
affinity for MEK1 (Table 2).
4
and 6 for MEK1 to that of 2,4-dihalogenated aniline
allosteric inhibitors such as cobimetinib revealed clear
differences with respect to binding interactions (for example,
an absence of contacts from 4 or 6 to K97 or ATP) (Figure
2
c), suggesting that compounds 4 and 6 represent novel and
differentiated allosteric MEK1 binders.
While we were unable to obtain crystal structures of all
fragment hits, the two we were able to obtain indicated clearly
that our screening method was indeed able to discover novel
allosteric MEK1 binders and encouraged us to also progress
Interestingly, removal of both chloro atoms (11) resulted in
a 3-fold increase in affinity, with further analogues strongly
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Table 2. Affinity and Efficiency Metrics of Analogues of 10
against MEK1
Table 3. Affinity and Efficiency Metrics of 4-Methoxyanilide
Derivatives against MEK1
a
Kd values are the geometric mean ± SEM of two or more
b
determinations. LE (kcal/(mol HA)): 1.37pK /HA (heavy atom
count). LipE: pK − log D. SPR pK in the presence of AMP-PNP
used in efficiency metrics. log D measured via shake-flask method in
d
c
d
d
d
e
octanol and water at pH 7.4. (μL/(min mg)). (μL/(min 1E6)).
f
−6
g
Papp: (*1 × 10 cm/s). n.d.: not determined. Compounds were
synthesized followed the synthetic route outlined in Scheme 1.
a
Kd values are the geometric mean ± SEM of two or more
b
determinations. LE (kcal/(mol HA)): 1.37pK /HA (heavy atom
count). LipE: pK − log D. SPR pK in the presence of AMP-PNP
used in efficiency metrics. log D measured via shake-flask method in
d
c
d
d
d
octanol and water at pH 7.4. n.d.: not determined.
indicating substitution with moderately bulky groups at the
ortho and meta positions to be detrimental to affinity for
MEK1 (12 and 13). Substitution of the para position, however,
appeared to result in favorable affinity gains for MEK1, with
fluoro (21), chloro (22), methoxy (14), and ethoxy (16)
substitutions all resulting in significant (up to 30-fold)
improvements in affinity. Larger substituents at the para
position were also tolerated (17 and 20), as were polar groups
such as phenols (19), although to a lesser degree.
Figure 4. (a) Crystal structure of MEK1 bound by compound 23.
Comparison of binding modes of 23 (b) and cobimetinib (c) for
MEK1. Parts b and c are shown in the same orientation (RMSD of
2
overlay: 1.092 Å for Cαs). The structure shown in part c is for PDB
1
entry 4AN2. AMP-PNP: adenylyl imidodiphosphate. AMP-PCP:
Focusing on the most ligand and lipophilic efficient
compound (14), the enantiomers 23 and 24 were separated,
and their absolute stereochemical configuration was assigned
by comparison of experimental and measured vibrational
circular dichroism (VCD) spectra (full details can be found in
the Supporting Information). The affinities for MEK1 were
assessed by SPR (Table 3), and this highlighted that the (S)
adenylyl methylenediphosphonate.
enantiomer (23) has a clear preference to bind MEK1 over its
antipode 24, with K values of 0.3 and 30 μM for 23 and 24,
d
respectively.
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With improved affinity, in vitro physicochemical and
ADMET properties of these compounds were then assessed
Table 3). Eutomer 23 was shown to possess good aqueous
scarcity of sulfur-containing MEK1 inhibitors may well be
intentional due to metabolic liabilities, high systemic
metabolism is a desirable property of an inhaled small-
molecule therapeutic. Therefore, 23 represents a promising
starting point for further optimization toward an inhaled
MEK1 therapeutic inhibitor.
(
solubility (355 μM), good permeability (as measured by the
Caco2 assay), and, encouragingly, no activity against the hERG
ion channel (IC50 > 40 μM). Furthermore, in vitro assessment
of metabolic stability in human liver microsomes (HLMs) and
rat hepatocytes (rHeps) indicated relatively low stability
compared to orally administered small molecules. For inhaled
administration, low metabolic stability may be advantageous,
We have reported here a fragment-based screening campaign
targeting MEK1 kinase which was designed to specifically
discover novel allosteric binders by screening with the ATP
binding site blocked. These efforts led to the discovery of
several novel allosteric MEK1 binders. These were confirmed
by X-ray crystal structures, and one series was optimized to
lead compound 23 with sub-μM binding affinity and promising
physicochemical and in vitro ADMET properties for the further
optimization toward an inhaled small-molecule therapeutic.
The same approach may likely be used to find Type III
inhibitors in few other kinases. The structural origin of the
existence of the allosteric pocket for binding of Type III kinase
inhibitors was studied in a recent paper by Zhao et al.
26,27
by limiting systemic exposure.
Early assessment of potential significant off-target inter-
actions was carried out on top compound 23 against a safety
panel (Eurofins SafetyScreen panel) of 22 off-targets,
comprising enzymes, receptors, and ion channels (e.g.,
COX2, PDEs, INSR, GABA, NMDA, CaV-L), as well as a
subpanel of 139 different kinases (Thermofisher kinome
subpanel). Compound 23 did not show any significant off-
target activity, with IC values >100 μM (SafetyScreen panel)
50
29
and percentage inhibitions <20% at 1.0 μM (kinome subpanel)
against all targets tested.
(
2017). The authors predicted that, apart from the MEK
proteins, this type of allosteric pocket might also exist in only
29,30
Having established promising affinity and ADMET proper-
ties, we next sought to understand the binding mode of
compound 23 to MEK1. Pleasingly, we were able to determine
a crystal structure of 23 in complex with MEK1 to a resolution
of 1.7 Å, revealing the compound to bind to the desired
allosteric site of the kinase (Figure 4a). The phenyl group
occupies a hydrophobic cavity formed by residues L118, F209,
V127, and M143. In this orientation, the ortho and meta
positions are sterically constrainedthe meta particularly so
correlating well with affinity data measured for the library
compounds 12 and 13 (Table 2). The methoxy group
protrudes into a smaller cavity, occupied by displaceable
water molecules, explaining the tolerance for slightly larger
substituents in this position. The oxothiomorpholine group is
positioned close to the imidodiphosphate group of AMP-PNP,
forming a hydrogen bond via its nitrogen to a phosphate
oxygen. 23 forms two further hydrogen bonds to the protein
one to the side chain of D208 and another to the side chain of
K97 (both catalytic residues). The hydrogen bond to D208,
formed from the linker amide NH of 23, appears to be a
particularly key contact, with the N-methyl variant of 23 (25)
essentially losing all binding affinity for the kinase. We note,
however, that steric clashes and/or conformational changes
may also contribute to the loss of activity of 25.
another 15 human kinases.
ASSOCIATED CONTENT
■
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00563.
Full details of all materials and methods used in this
work including protein production, SPR, NMR, X-ray
crystallography, VCD, and chemical synthesis (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
Paolo Di Fruscia − Structure Biophysics & Fragments,
Discovery Sciences, R&D, AstraZeneca, Cambridge CB4
0
WG, United Kingdom; orcid.org/0000-0002-2914-
0651; Email: paolo.difruscia@astrazeneca.com
Fredrik Edfeldt − Structure Biophysics & Fragments,
Discovery Sciences, R&D, AstraZeneca, Gothenburg 431 83,
Sweden; Email: fredrik.edfeldt@astrazeneca.com
̈
Helena Kack − Structure Biophysics & Fragments, Discovery
Sciences, R&D, AstraZeneca, Gothenburg 431 83, Sweden;
orcid.org/0000-0002-5456-3269; Email: helena.kack@
astrazeneca.com
Comparison of the binding mode of 23 to MEK1 to that of
cobimetinib shows that the methoxy-phenyl group of 23
occupies the same position as the 2,4-dihalogen group of
cobimetinib. Polar contacts to K97 and the terminal phosphate
of the bound ATP analogue also appear to be a shared feature
of both compounds; however, the hydrogen bond formed from
Authors
Igor Shamovsky − Medicinal Chemistry, Research & Early
Development, Respiratory & Immunology,
2
3 to the catalytic residue D208 does not feature in the
BioPharmaceuticals R&D, AstraZeneca, Gothenburg 431 83,
Sweden; orcid.org/0000-0002-2881-9531
Gavin W. Collie − Structure Biophysics & Fragments,
Discovery Sciences, R&D, AstraZeneca, Cambridge CB4
0WG, United Kingdom; orcid.org/0000-0002-0406-
922X
Anna Aagaard − Structure Biophysics & Fragments, Discovery
Sciences, R&D, AstraZeneca, Gothenburg 431 83, Sweden
Louise Barlind − Discovery Biology, Discovery Sciences, R&D,
AstraZeneca, Gothenburg 431 83, Sweden
binding mode of cobimetinib (Figure 4c). A broader
inspection of available crystal structures of MEK1 bound by
allosteric inhibitors indicates interactions with D208 to be rare.
Targeting conserved catalytic residues is of particular interest
as such residues would be expected to be less likely to be the
site of acquired-resistance mutation in oncology settings due to
their crucial enzymatic function. In addition, the presence of
electron-rich sulfur atoms within allosteric MEK1 inhibitors
also appears rare, and we were able to identify few examples of
such compounds bound to MEK1 in the Protein Data Bank
Ulf Borjesson − Structure Biophysics & Fragments, Discovery
̈
Sciences, R&D, AstraZeneca, Gothenburg 431 83, Sweden
22
28
(
e.g., PDB entries 3EQH and 3SLS ). Indeed, while the
3
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Note
Eva L. Hansson − Mechanistic Biology and Profiling,
Discovery Sciences, R&D, AstraZeneca, Gothenburg 431 83,
Sweden
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5) Caunt, C. J.; Sale, M. J.; Smith, P. D.; Cook, S. J. MEK1 and
Richard J. Lewis − Medicinal Chemistry, Research & Early
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BioPharmaceuticals R&D, AstraZeneca, Gothenburg 431 83,
Sweden; orcid.org/0000-0001-9404-8520
Magnus K. Nilsson − Medicinal Chemistry, Research & Early
Development, Respiratory & Immunology,
BioPharmaceuticals R&D, AstraZeneca, Gothenburg 431 83,
Sweden
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Linda Oster − Structure Biophysics & Fragments, Discovery
Sciences, R&D, AstraZeneca, Gothenburg 431 83, Sweden
Josefine Pemberton − Structure Biophysics & Fragments,
Discovery Sciences, R&D, AstraZeneca, Gothenburg 431 83,
Sweden
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Vollmer, S.; Baturcam, E.; Svanberg, P.; Bonn, B.; Smith, P. D.;
Vaarala, O.; Cunoosamy, D. M. Dual Role For A MEK Inhibitor As A
Modulator Of Inflammation And Host Defense Mechanisms With
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Lena Ripa − Medicinal Chemistry, Research & Early
Development, Respiratory & Immunology,
BioPharmaceuticals R&D, AstraZeneca, Gothenburg 431 83,
Sweden; orcid.org/0000-0003-1533-1673
R. Ian Storer − Structure Biophysics & Fragments, Discovery
Sciences, R&D, AstraZeneca, Cambridge CB4 0WG, United
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Inhibits LPS-Induced TNFα Production through MEK/ERK
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based lead discovery (FBLD). Essays Biochem. 2017, 61, 453−464.
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00563
(
J. C.; Jazayeri, A.; Marshall, F. H.; Okrasa, K.; Serrano-Vega, M. J.;
Tehan, B. G.; Wiggin, G. R.; Congreve, M. Fragment and Structure-
Based Drug Discovery for a Class C GPCR: Discovery of the mGlu5
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ropyridin-2-yl)pyrimidin-4-yl]benzonitrile. J. Med. Chem. 2015, 58,
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
6
(
653−6664.
The authors declare no competing financial interest.
Accession Codes: Atomic coordinates and structure factors
have been deposited in the Protein Data Bank with accession
codes 7B7R (4), 7B3M (6), 7B94 (9), and 7B9L (23).
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Approaches for Allosteric Metabotropic Glutamate Receptor
ACKNOWLEDGMENTS
■
(
mGluR) Modulators. Curr. Top. Med. Chem. 2019, 19, 1768−1781.
We thank Pharmaron China for supporting the synthetic work.
(15) Scott, D. E.; Coyne, A. G.; Hudson, S. A.; Abell, C. Fragment-
based approaches in drug discovery and chemical biology.
Biochemistry 2012, 51, 4990−5003.
ABBREVIATIONS
■
(16) Tiefenbrunn, T.; Forli, S.; Happer, M.; Gonzalez, A.; Tsai, Y.;
SPR, surface plasmon resonance; COPD, chronic obstructive
pulmonary disease; MEK1, dual specificity mitogen-activated
protein kinase kinase 1; AMP-PNP, adenylyl-imidodiphos-
phate; FBDD, fragment-based drug discovery; DMF, N,N-
dimethylformamide; HATU, hexafluorophosphate azabenzo-
triazole tetramethyl uronium; DIPEA, N,N-diisopropylethyl-
amine.
Soltis, M.; Elder, J. H.; Olson, A. J.; Stout, C. D. Crystallographic
fragment-based drug discovery: use of a brominated fragment library
targeting HIV protease. Chem. Biol. Drug Des. 2014, 83, 141−148.
(17) Vance, N. R.; Gakhar, L.; Spies, M. A. Allosteric Tuning of
Caspase-7: A Fragment-Based Drug Discovery Approach. Angew.
Chem., Int. Ed. 2017, 56, 14443−14447.
(18) Li, C.; Zhang, X.; Zhang, N.; Zhou, Y.; Sun, G.; Zhao, L.;
Zhong, R. Identification and Biological Evaluation of CK2 Allosteric
Fragments through Structure-Based Virtual Screening. Molecules
2020, 25, 237.
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(
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