Design, Synthesis and Characterization of Covalent KDM5 Inhibitors

Article abstract of DOI:10.1002/anie.201810179

Histone lysine demethylases (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub-family. The covalent binding to the targeted proteins was confirmed by MS and time-dependent inhibition. Additional competition assays show that compounds were non 2-OG competitive. Target engagement and ChIP-seq analysis showed that the compounds inhibited the KDM5 members in cells at nano- to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites.

Full text of DOI:10.1002/anie.201810179

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Design, Synthesis and Characterization of Covalent KDM5
Inhibitors
Authors: Saleta Vazquez-Rodriguez, Miranda Wright, Catherine M.
Rogers, Adam Cribbs, Srikannathasan Velupillai, Martin
Philpott, Henry Lee, James E Dunford, Kilian V. M. Huber,
Mathew B. Robers, James D. Vasta, Marie Laetitia Thezenas,
Sarah Bonham, Benedikt Kessler, James Bennett, Oleg
Fedorov, Florence Raynaud, Adam Donovan, Julian Blagg,
Vassilios Bavetsias, Udo Oppermann, Chas Bountra, Akane
Kawamura, and Paul Brennan
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201810179
Angew. Chem. 10.1002/ange.201810179
Link to VoR: http://dx.doi.org/10.1002/anie.201810179
http://dx.doi.org/10.1002/ange.201810179

10.1002/anie.201810179
Angewandte Chemie International Edition
COMMUNICATION
Design, Synthesis and Characterization of Covalent KDM5
Inhibitors
Saleta Vazquez-Rodriguez,+ Miranda Wright,+ Catherine M. Rogers, Adam Cribbs, Srikannathasan
Velupillai, Martin Philpott, Henry Lee, James E. Dunford, Kilian V. M. Huber, Matthew B. Robers,
James D. Vasta, Marie-Laetitia Thezenas, Sarah Bonham, Benedikt Kessler, James Bennett, Oleg
Fedorov, Florence Raynaud, Adam Donovan, Julian Blagg, Vassilios Bavetsias, Udo Oppermann,
Chas Bountra, Akane Kawamura,* Paul E. Brennan*
Abstract: Histone lysine demethylase (KDMs) are involved in the
dynamic regulation of gene expression and they play a critical role in
several biological processes. Achieving selectivity over the different
KDMs has been a major challenge for KDM inhibitor development.
Here we report potent and selective KDM5 covalent inhibitors
designed to target cysteine residues only present in the KDM5 sub-
family. The covalent binding to the targeted proteins was confirmed
by MS and time-dependent inhibition. Additional competition assays
show that compounds were non 2-OG competitive. Target
engagement and ChIP-seq analysis showed that the compounds
inhibited the KDM5 members in cells at nano- to micromolar levels
and induce a global increase of the H3K4me3 mark at transcriptional
start sites.
methyltransferases (HMTs) and lysine demethylases (KDMs)
that control the methylation state (mono- di- or tri-methylation) of
lysine residues in histone tails.[1] The larger class of KDMs, the
Jumonji-C domain containing lysine demethylases (JmjC-KDMs)
utilize Fe(II) and 2-oxoglutarate (2-OG) to oxidise methylated
lysine substrates to form an unstable hemiaminal which
fragments to the demethylated lysine and formaldehyde.[2]
JmjC-KDMs are a family of 20 human enzymes which are
further categorized into subfamilies KDM2-7.[3] There are four
enzymes in the KDM5 subfamily (KDM5A-D) which can all
demethylate di- and tri-methylated lysine
4 of histone 3
(H3K4me2/3), ubiquitous histone marks necessary for
transcriptional activation. Various studies have linked the
KDM5s to different cancers, in particular overexpression of
KDM5B has been observed in prostate, gastric, breast, ovarian
Histone proteins can be covalently modified by a plethora of
and hepatic cancer cells.[4] KDM5B also has
a role in
post-translational
marks
including
methylation.
These
modifications modulate chromatin structure which impacts gene
expression, chromosome packaging and DNA damage repair.
development by blocking differentiation of embryonic and
hematopoietic stem cells.[5] the KDM5s have therefore become
increasingly popular targets for chemical probe and drug
discovery to gain a greater understanding of their role in biology
and as oncology targets.
Methylation is
a reversible process regulated by histone
[*]
Dr. S. Vazquez-Rodriguez, Dr. M. Wright, Dr. C. M. Rogers, , Dr. S.
Velupillai , Dr. K. V. M. HuberJ. Bennett, Dr. O. Fedorov, Prof. Dr. U.
Oppermann, Prof. Dr. C. Bountra and Prof. Dr P. E. Brennan
Structural Genomics Consortium & Target Discovery Institute
University of Oxford, NDM Research Building
Several small-molecule inhibitors of have been reported.[6]
They bind to KDM5s through coordination to the active site Fe(II)
in the 2-OG binding site and form a salt bridge with a conserved
lysine or hydrogen bond to a conserved tyrosine residue in the
KDM5 subfamily. Although nanomolar enzyme IC50’s are
reported, cellular activities are commonly much weaker which
has been attributed to competition of the inhibitors with higher
levels of competing substrate in cells (2-OG ~1 mM)[7] compared
to that used in biochemical assay conditions (low µM range), a
factor known to reduce kinase inhibitor efficacy.[8] Although
KDM5 inhibitors have shown good selectivity over KDM2/3/6,
selectivity over the KDM4 subfamily has proven more
challenging as the primary binding residues of the catalytic
domain are conserved in KDM4 and KDM5. Selectivity has only
been achieved through structural changes in the secondary shell.
For example, cyanopyrazole CPI-455 is a potent, selective
KDM5 inhibitor with nanomolar enzyme IC50, but micromolar
cellular IC50.[9] Further optimisation led to tert-butyl pyrazole 1[10]
which showed improved cellular activity (Figure 1B). Potent
Roosevelt Drive, Oxford, OX3 7DQ and OX3 7FZ (UK)
E-mail: paul.brennan@sgc.ox.ac.uk
Dr. A. Cribbs, Dr. M. Philpott, H. Lee, Dr. J. E. Dunford and Prof. Dr.
U. Oppermann
Botnar Research Center, Nuffield Department of Orthopedics,
Rheumatology and Musculoskeletal Sciences
NIHR Oxford BRC, University of Oxford, OX3 7DQ, UK
Prof. Prof. Dr. U. Oppermann
FRIAS - Freiburg Institute of Advanced Studies, 79104, Freiburg,
Germany
Dr. A. Kawamura and Dr. M. Wright
Chemistry Research Laboratory, University of Oxford,
12 Mansfield Road, Oxford, OX1 3TA (UK)
Dr. M. B. Robers and Dr. J. D. Vasta
Promega Corporation
2800 Woods Hollow Road, Fitchburg,
Wisconsin 53711, USA
M. L. Thezenas, Dr. S. Bonham and Prof. Dr. B. Kessler
Target Discovery Institute, Nuffield Department of Medicine,
University of Oxford, Roosevelt Drive, 7FZ Oxford, UK
KDM5 inhibitors, such as
2, incorporating
a
8-
F. Raynaud, A. Donovan, Prof. Dr. Julian Blagg, Dr. Vassilios
Bavetsias
Cancer Research UK Cancer Therapeutics Unit,
The Institute of Cancer Research,
pyrazolopyridopyrimidinone core were published, however this
series suffered from poor selectivity over KDM4 and poor
cellular potency despite cellular permeability.[11]
15 Cotswold Road, London SM2 5NG, U.K
Here we report two series of KDM5 selective covalent
[+] These authors contributed equally to this work.
inhibitors
based
on
cyanopyrazole
CPI-455
and
Supporting information for this article is given via a link at the end of
the document
pyridopyrimidinone 2 with improved selectivity that target key
cysteines exclusive to the KDM5s. Irreversible binding of the
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10.1002/anie.201810179
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protein to the inhibitor resulted in reduced competition with the
2-OG cosubstrate and maintained their cellular activity despite
low cellular permeability. Covalent inhibitors are potentially more
selective and potent, with better cellular efficiency and can
require less frequent and lower doses, although this can be
offset by poor selectivity with the cellular cysteinome. Covalent
inhibitors can also be used as proteomic tools for both covalent
and non-covalent drugs.[12]
Pyridopyrimidinone-based covalent inhibitors 3-6 (PP series)
(Figure 1D) were synthesised (Schemes S1-S2) and acrylamide
3 was co-crystallised with KDM5B to determine the binding
mode. Bidentate metal coordination and salt bridging/hydrogen
bonding to K517/Y425 was observed with inhibitor 3 and a
covalent bond was seen between the C480 sulfur and
acrylamide β-carbon (Figure 1C).
A second series of KDM5 covalent inhibitors 7-11 (PZ series)
(Figure 1E) were also designed and synthesized (Schemes S3-
S4) to target C497. X-ray crystal structures of 7 and 10 showed
the reactive chloroacetyl and acrylamide moieties within
proximity of C497 sulfur (7, 5.8 Å; 10, 11.7 Å) (Figure S1)
although no covalent bond was seen in the crystal structure. The
acrylamide of 10 was positioned further from C497 however
compound flexibility could position it within covalent bonding
distance. Both compounds maintained the key coordination of
the nitrile to the metal centre and h-bonding interactions of the
carbonyl oxygen.
Based on alignments of the JmjC-KDMs, two cysteine
residues were identified as potential nucleophiles for covalent
modifications in the KDM5 family. C497 in KDM5B was identified
as a non-catalytic residue close to the binding site present only
in the KDM5 members. In addition, C480 was identified as a
unique residue in KDM5B, so improved selectivity could be
achieved over KDM4s as well as KDM5A/C/D (Figure 1A). The
irreversible binding of the covalent inhibitors to KDM5s could
also reduce competition with 2-OG to improve their cellular
Since covalent inhibitors possess time-dependent inhibition
due to the kinetics of covalent binding to the protein, their activity
was better assessed through determination of the kinetic
parameter kinact/Ki, rather than a simple IC50. These parameters
were calculated by using an established method to derive Ki and
kinact directly from time-dependant IC50’s (Table S1). IC50 values
were measured using an AlphaScreen assay[13] by pre-
incubating the compounds with KDM5B at different time points
ranging from 0 to 120 minutes and the kinact and Ki values
determined as previously described.[14]
In the PP series, chloroacetamide 5 was the most potent with
the highest kinact/Ki (40 x 103/Ms) and although acrylamide 4 was
less active than 5 with (kinact/Ki 7.4 x 103/Ms) it showed a drop in
IC50 of 12-fold after an hour incubation (Figure 2A).
Dimethylamino crotonamide
6 was less potent than the
acrylamide 4 with a kinact/Ki of 6.1 x 103/Ms. Acrylamide 4 was 5-
fold more potent than the corresponding piperazine acrylamide 3
(kinact/Ki 1.4 x 103/Ms).
Figure 2 A) Time-dependent inhibition of covalent inhibitors 4 and 7; B-C)
2-OG competition assay with non-covalent variants (1 and 2) and covalent
inhibitors (4 and 7).
In the PZ series, chloroacetamide 7 showed the greatest
kinact/Ki (25 x 103/Ms) with an IC50 drop of 27-fold upon pre-
incubation (Figure 2A). Both acrylamide 10 and chloroacetamide
11 had comparable kinact/Ki values of 20 x 103 and 19 x 103/Ms,
while the phenyl analogues 8 and 9 showed the lowest kinact/Ki
ratio with 6.9 x 103 and 5.8 x 103/Ms, respectively. The larger
drop in IC50 with the PZs compared to the PPs was likely due to
the greater contribution of covalency to their potency. Overall,
activity.
Figure 1. A) KDM alignment at positions C480 and C497 (KDM5B
numbering); B) Reported KDM5 inhibitors CPI-455, 1 and 2; C) Overlay of
compound 2 docked in the X-ray structure of 3 (green) in KDM5B (PDB ID
6EIN); B) PP compounds; D) PZ compounds.
C480 is positioned close to the binding site of previously
reported inhibitor[11] 2 and was suitable for the design of
cysteine-selective electrophiles based on this scaffold. 8-
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10.1002/anie.201810179
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COMMUNICATION
compounds in both series possessed time-dependent inhibition
through covalent binding to KDM5B leading to potent nanomolar
IC50 values.
When comparing the activity of the compounds in the KDM5
family members, both series were equipotent against
KDM5A/B/D, but 30–100-fold selective over KDM5C. In the PP
series, compound 6 was the most selective with greater than 7-
fold selectivity for KDM5B over KDM5A/C/D which could be
explained by specific binding to C480. In the PZ series, the most
selective compound was chloroacetamide 11 showing the
highest selectivity for KDM5B with more than 100-fold over
KDM5C.
As well as improving their potency, the addition of a covalent
electrophile to the inhibitors was expected to reduce competition
with 2-OG. IC50 values of both non-covalent (1 and 2) and
covalent inhibitors (4 and 7) were therefore determined at
different concentrations of 2-OG up to 1000 µM (Figure 2B-C).
In the PP series, the non-covalent analogue 2 showed an
approximately 500-fold drop in potency from 0.5 µM up to 1000
µM while the covalent inhibitor 4 showed only an 18-fold lower
potency at the high 2-OG concentration. At 1 mM 2-OG, 4 was
4-fold more potent with an IC50 of 110 nM than 2 which we
attribute to improved competition from 2-OG compared to 2 by a
factor of 25.
As reported,[9] even the non-covalent cyanopyrazoles CPI-455
and 1 are less competed by 2-OG than the PP series, with
compound 1 showing a drop in potency of only 8-fold in the
range of 2-OG concentrations measured. As PZ compound 1
and PP compound 2 have similar binding modes based on their
crystal structures, it is not clear why they show such different
competition behaviour. But the covalent PZ inhibitor 7 was
significantly more potent than non-covalent PZ 1 with an IC50 at
the top 2-OG concentration of 10 nM and showed only a small
shift in potency of 4-fold with 2-OG concentration. The addition
of covalent inhibition to the PZs improved the potency as well as
the competition with 2-OG. Covalent inhibitors therefore
represent an excellent strategy for solving the issues with KDM
inhibitors in which high 2-OG competition could explain their
poor cellular activities.
Covalent binding of the inhibitors with KDM5B was confirmed
through MS-labelling experiments (Figure 3). PP compound 3
was incubated with KDM5B and the resulting MS spectra
showed a minor set of peaks relating to unmodified KDM5B (m/z
55,153) and a major peak relating to protein-inhibitor adduct with
the correct mass shift (Δm/z 379) (Figure 3A). Compounds 4-6
were incubated with KDM4B and KDM5C under the same
conditions as with KDM5B (Figure S2) and MS showed only
unmodified protein peaks as expected for these proteins that do
not contain a cysteine at C480 as in KDM5B. The specific
covalent binding to KDM5B explains the improved selectivity
over KDM5C and KDM4B compared to non-covalent inhibitor 2.
Digestion of the protein-inhibitor adduct and further analysis
by LCMS/MS allowed mapping of the inhibitor reactive residue,
confirming that compound 3 was binding specifically to the
desired C480 and not to other cysteines in KDM5B (Figure 3B,
green box) in agreement with X-ray crystal structure (Figure 1C).
Similarly, incubation of 7 with KDM5B resulted in protein MS
peaks from addition of the compound (Δm/z 351) (Figure 3A).
LCMS/MS analysis (Figure 3B) showed that although there was
good coverage of the native protein (>90%) including 3 peptides
containing C497, with the inhibitor modified KDM5B, the peptide
containing the target C497 was the only sequence that was not
observed (Figure 3B light blue box). As this peptide was
observed in the unmodified protein, the addition of the
compound appears to have prevented the detection of the
peptide in the covalent adduct suggesting that C497 was the site
of binding. Compounds 4, 7 and 9 were also incubated with
PCAF[15] and NUDT7 (PDB ID 5QHH), two enzymes known to
have covalent inhibitors, but no labelling was observed,
demonstrating the selectivity for KDM5B (Figure S3).
A panel of JmjC KDMs was tested by AlphaScreen to
determine the selectivity of the compounds (Table S2). IC50’s
were measured at 120 minutes when the inhibition of the
covalent inhibitors against KDM5B had levelled off (Figure 2A).
All the compounds from both series presented selectivity
towards KDM5s, with the highest potency for the most closely
related KDM5A and 5B. For the PZ series, the most potent
compounds against KDM5B contained the 2-chloroacetamide
group, with 7 being the most potent of the PZ series (KDM5A/B
IC50 10 nM) and showed the best selectivity profile (Table S3). In
the PP series the covalent compounds tested showed high
activity with IC50 in the low nanomolar range against most KDM5
family members.
Both PP and PZ series showed the greatest selectivity over
KDM2A and 3A with selectivity from 200- to 1500-fold. The
covalent inhibitors also demonstrated good selectivity over the
most closely related KDM4 subfamily members. In the PP series,
methylbenzylamine covalent inhibitors 4-6 showed greater than
20-fold selectivity over the KDM4 family, in particular 5 was
more than 60-fold less active on KDM4B. We attribute this
selectivity for KDM5B with the covalent PP inhibitors to binding
to C480. In the PZ series, all compounds were more than 50-fold
selective over all the KDM4 family members, with compounds 7
and 10 having >500-fold selectivity over KDM4A/B.
Figure 3. A) Intact mass spectra of covalent binding by MS; B) Peptide
mapping of KDM5B after treatment with compounds 3 and 7. Observed
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10.1002/anie.201810179
Angewandte Chemie International Edition
COMMUNICATION
10]
[d] AlphaScreen in vitro activity in KDM5B. [e] Calculated as ratio of IC50’s
peptides for native protein ( light red box), compound 3 adduct (green box)
and compound 7 adduct (light blue box).
determined at 2-OG concentrations of 1 mM and 1 µM.
Immunofluorescence-based assays have been previously
used to analyse global changes in H3K4me3 as a measure of
KDM5 activity, but the global change can be affected by other
factors such as cytotoxicity.[17] We therefore employed chromatin
immunoprecipitation and sequencing (ChIP-seq) as a more
accurate method to quantify the H3K4me3 level at
transcriptional start sites (TSS) as a read-out for inhibition of
KDM5 activity. Cytotoxicity of the compounds was first assessed
to show that with the exception of 5, compounds were not
cytotoxic in HEK cells (Figure S7). Compounds 4, 7 and 9 were
With confirmation of potent and selective binding to
recombinant KDM5 targets, we wanted to show cellular target
engagement. We developed
a
PZ-based cell-permeable
fluorescent tracer assay to quantify the engagement of
compounds with KDM targets that are fused to
NanoLuciferase.[16] An aliphatic amino-terminal linker on a PZ
derivative was attached to the fluorescent dye NanoBRET590® to
generate a tracer compound that interacts with the NanoLuc-
fused KDMs (Figure S4).
The tracer was tested in transfected NanoLuc-KDM4s, KDM5s
and KDM6B in the presence of compound 1, showing that the
tracer produced significant BRET signal in a dose-dependent
manner with all the KDM5 family members (KDM5A-D), but no
BRET signal for any of the KDM4s or KDM6B, demonstrating
the selectivity of the tracer (Figure S5). Cellular activity of the
most relevant PP and PZ compounds was then evaluated (Table
1, Figure 4A, Figure S6). In the PZ series, phenyl-containing
compounds 8 and 9, showed the best activity in cells (0.53 and
0.30 μM). However, compounds 7, 10 and 11 showed
micromolar IC50 values, which are more than a 100-fold weaker
than their in vitro activity, probably due to their low logP and
poor cell permeability.
selected
for
H3K4me3
ChIP-seq
experiments
and
quantification.[18]
Figure 4. A) Cellular activity of compound 9 in by NanoBRET assay; B)
Distribution of H3K4me3 around transcriptional start sites (TSS); Densities of
ChIP-seq reads for H3K4me3 and input in HEK293 cells treated with DMSO
and compounds 4, 7, 9 and non-covalent reference compound KDOAM-25.
After sequencing and normalisation, read coverage was
evaluated and coverage plots were plotted across all genes
revealing significant increases of H3K4me3 levels around TSS
(Figure 4B). ARID3B, known to be highly expressed in HEK
cells,[19] was chosen as a representative gene and all three
compounds doubled H3K4me3 levels (Figure S8).
Compound 4 was the only example of the PP series that
showed some activity in cells, with an IC50 of 10.6 μM, in the
same range as the non-covalent compound 2 (IC50 8.8 μM). For
both the PP and PZ compounds, the similar cellular potency
compared to their non-covalent analogues can be explained due
to their high polarity as measured clogP and poor cell
permeability as measured by Caco-2 (Table 1). Non-covalent
compounds CPI-455, 2 and 3 have good permeability (Caco-2
AB 7.5–17.2 x 10-6 cm/s) but despite being much less cell
In conclusion, covalent inhibitors in the PP and PZ series that
target distinctive cysteine residues in the KDM5 family has been
shown to target lysine demethylases to overcome high cellular
2-OG levels and improve KDM selectivity. Target engagement in
cells was demonstrated using a novel NanoBRET assay and
functional effects of the covalent inhibitors was shown by
increase in H3K4me3 at TSS in HEK293 cells.
permeable (Caco-2 AB <1.5
x
10-6 cm/s), the covalent
compounds are as potent in cells as their non-covalent
analogues, presumably due to their better 2-OG competition.
Table 1. Biochemical and cellular properties of the PZ and PP series
IC50
IC50
KDM5B
nano-
Cmpd
(2-OG (2-OG 2-OG
ChIP-seq
conc
(µM)
clogP
Papp
1 µM) 1 mM)
shift
BRET
IC50 (µM)
[a]
[b]
[d]
[d]
[e]
Acknowledgements
2
3
4
5
6
1.1
-1.8
11.8
<1.5
<1.5
<1.5
<1.5
0.003
0.184
0.009
0.01
0.506
ND
169
ND
ND
11
8.8
>30
10.6
>30
>30
ND
ND
ND
2.0
ND
The SGC is a charity (number 1097737) funded by AbbVie,
Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation
for Innovation, Eshelman Institute for Innovation, Genome
Canada, Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD
grant no. 115766], Janssen, Merck KGaA Darmstadt, MSD,
Novartis Pharma AG, Ontario Ministry of Economic
Development and Innovation, Pfizer, São Paulo Research
Foundation-FAPESP, Takeda, and Wellcome [106169/ZZ14/Z].
Funding is from the European Union’s Horizon 2020 research
and innovation programme under the Marie Sklodowska-Curie
grant agreement No 744389, supporting S.V.R. fellowship
(TEDCIP). M.W. was supported by the EPSRC Centre for
Doctoral Training in Synthesis for Biology and Medicine
(EP/L015838/1). Funding was received from the Oxford NIHR
-1.5
ND
-0.94
-0.58
0.11
0.094
0.004
26
CPI-
455
1.3
17.2[c] 0.003[c]
ND
2.0[c]
0.40
ND
1
7
0.65
-0.74
0.68
0.92
-0.89
-1.3
7.5[c]
<0.76
<0.76
<0.76
<0.76
<1.4
0.015
0.007
0.049
0.065
0.017
0.009
0.377
0.010
0.041
0.059
0.017
0.008
3.9
2.0
1.8
1.9
2.4
1.3
0.34
2.5
ND
2.0
2.0
ND
ND
ND
8
0.53
0.30
5.5
9
10
11
8.6
[a] Calculated with ChemDraw version 16.0.1.4. [b] Determined in Caco-2 cells.
Values are A to B (10-6 cm/s). [c] Previously reported values in MDCK cells.[9-
This article is protected by copyright. All rights reserved.

10.1002/anie.201810179
Angewandte Chemie International Edition
COMMUNICATION
[7]
K. Thirstrup, S. Christensen, H. A. Moller, A. Ritzen, A. L.
BRC, Arthritis Research UK (grant 20522) and the People
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Framework Programme (FP7/2007-2013) (UO) under REA grant
n° [609305].
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268-273.
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[9]
[10]
[11]
[12]
Keywords: covalent inhibitors • epigenetics • KDM5 • lysine
demethylase
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