Selectively Targeting the Kinome-Conserved Lysine of PI3Kδ as a General Approach to Covalent Kinase Inhibition

Article abstract of DOI:10.1021/jacs.7b08979

Selective covalent inhibition of kinases by targeting poorly conserved cysteines has proven highly fruitful to date in the development of chemical probes and approved drugs. However, this approach is limited to 200 kinases possessing such a cysteine near the ATP-binding pocket. Herein, we report a novel approach to achieve selective, irreversible kinase inhibition, by targeting the conserved catalytic lysine residue. We have illustrated our approach by developing selective, covalent PI3Kδ inhibitors that exhibit nanomolar potency in cellular assays, and a duration of action >48 h in CD4+ T cells. Despite conservation of the lysine residue throughout the kinome, the lead compound shows high levels of selectivity over a selection of lipid and protein kinases in biochemical assays, as well as covalent binding to very few off-target proteins in live-cell proteomic studies. We anticipate this approach could offer a general strategy, as an alternative to targeting non-conserved cysteines, for the development of selective covalent kinase inhibitors.

Full text of DOI:10.1021/jacs.7b08979

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Article
Selectively targeting the kinome-conserved lysine of
PI3K# as a general approach to covalent kinase inhibition
Samuel E Dalton, Lars Dittus, Daniel A. Thomas, Maire A. Convery, Joao Nunes, Jacob T. Bush,
John P. Evans, Thilo Werner, Marcus Bantscheff, John A. Murphy, and Sebastien Campos
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b08979 • Publication Date (Web): 12 Dec 2017
Downloaded from http://pubs.acs.org on December 12, 2017
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Selectively targeting the kinome-conserved lysine of PI3Kδ as a gen-
eral approach to covalent kinase inhibition
Samuel E Dalton,†,‡ Lars Dittus,¥ Daniel A Thomas,‡ Máire A Convery,‡ Joao Nunes,‡ Jacob T
Bush,‡ John P Evans,‡ Thilo Werner,¥ Marcus Bantscheff,¥ John A Murphy,† and Sebastien Cam-
pos‡,*
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† Department of Pure and Applied Chemistry, WestCHEM, University of Strathclyde, 295 Cathedral Street, Glasgow,
G1 1XL, U.K.
‡ GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, U.K.
¥ Cellzome GmbH, a GSK company, Meyerhofstr. 1, Heidelberg, 69117, Germany
ABSTRACT: Selective covalent inhibition of kinases by targeting poorly conserved cysteines has proven highly fruitful to
date in the development of chemical probes and approved drugs. However, this approach is limited to ~200 kinases pos-
sessing such a cysteine near the ATP-binding pocket. Herein, we report a novel approach to achieve selective, irreversible
kinase inhibition, by targeting the conserved catalytic lysine residue. We have illustrated our approach by developing se-
lective, covalent PI3Kδ inhibitors that exhibit nanomolar potency in cellular assays, and a duration of action >48 h in
CD4+ T cells. Despite conservation of the lysine residue throughout the kinome, the lead compound shows high levels of
selectivity over a selection of lipid and protein kinases in biochemical assays, as well as covalent binding to very few off-
target proteins in live-cell proteomic studies. We anticipate this approach could offer an alternative general strategy, to
targeting non-conserved cysteines, for the development of selective covalent kinase inhibitors.
INTRODUCTION
variety of diseases.21,22 A number of selective reversible
PI3Kδ small molecule inhibitors have entered clinical tri-
als, with Zydelig recently obtaining FDA approval as a
second-line treatment for relapsed follicular B-cell non-
Hodgkin lymphoma, and relapsed chronic lymphocytic
leukemia.23–25 More recently, drug developers have target-
ed PI3Kδ for the treatment of inflammatory conditions
such as asthma, chronic obstructive pulmonary disease
(COPD), rheumatoid arthritis and activated PI3Kδ syn-
drome.21,26–32
The clinical successes of ibrutinib1 and afatinib2 have
prompted a resurgence of interest in covalent drug dis-
covery.3,4 Covalent inhibitors can possess the advantages
of increased potency, prolonged duration of action, de-
coupled pharmacodynamics and pharmacokinetics, and,
often, require less frequent and lower doses.4,5
In the kinase field, researchers commonly target cyste-
ine residues for covalent inhibition.6 Targeted residues
are often “non-catalytic and poorly conserved”7 to maxim-
ise selectivity, and mitigate the risk of off-target covalent
interactions.4,5,7,8 However, only ~200 of over 500 human
kinases have been mapped with a cysteine in the vicinity
of the ATP pocket, and <50 have been demonstrated to
covalently engage with inhibitors, restricting the scope of
this strategy.6,9–11 We chose to challenge this general ap-
proach by investigating the potential to selectively and
irreversibly target the kinome-conserved lysine residue.12
Covalent conjugation with lysine is far less common, due
to its protonation state, and therefore poorer nucleo-
philicity under physiological conditions.13 Nonetheless,
interest in this nucleophile is rapidly gaining traction in
the scientific community.14–18
To our knowledge, a selective covalent PI3Kδ inhibitor
has not yet been disclosed, and there is no obvious iso-
form specific nucleophilic residue to target around the
ATP binding site. Irreversible pan-PI3K inhibitors have
been investigated previously, based on the fungal antibi-
otic wortmannin.33–35 These compounds target the con-
served lysine12,36,37 for the covalent reaction, but are poorly
selective.35 Promiscuous kinase probes that covalently
bind to this residue,38–41 have also been developed and
commercialised, however methods of selectively targeting
this lysine in specific kinases have not yet been reported.
Herein we describe the development of the first selec-
tive, irreversible PI3Kδ inhibitor, which reacts with the
conserved catalytic lysine (Lys779 in PI3Kδ numbering).
Selectivity was achieved through optimisation of the re-
versible interactions in formation of the initial enzyme-
The heterodimeric lipid kinase phosphoinositide 3-
kinase delta (PI3Kδ)19,20 has been targeted specifically over
the related PI3Kα, β and γ isoforms for the treatment of a
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inhibitor complex, whilst the rate of the covalent reaction
with the protein remained constant. The targeted lysine
residue is present throughout the kinome, hence we an-
ticipate that this strategy could provide an alternative
general approach for the development of selective irre-
versible kinase inhibitors.
Page 2 of 9
sis, with biochemical potencies on-par with wortmannin
10, and selectivity comparable to the FDA-approved
PI3Kδ drug, Zydelig24 11. Furthermore, compounds 3-5
provided good levels of inhibitory activity in the hWB
assay (~10 nM), supporting engagement of PI3Kδ in cells,
and were at least 15-fold more potent than 10 and 11 in
this assay. It is worth noting that covalent inhibition is a
time-dependent process, and the pIC50 values would be
expected to vary with time. The biochemical assays were
read-out at 1 h in all cases, and the hWB assay at 20 h to
provide consistency for data analysis.
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RESULTS AND DISCUSSION
Design of lysine targeting inhibitors. Clinical candi-
date 1 was identified as a suitable starting point due to its
potency profile, and reversible interaction between the
sulfonamide and Lys779.23 We replaced the cis-
dimethylmorpholine with the more basic, more soluble
piperazine moiety23 and hypothesised that substitution of
the sulfonamide for an electrophilic functional group
could afford covalent inhibitors (Figure 1). In silico mod-
elling suggested that activated esters42 would be tolerated
in a reversible enzyme-inhibitor complex with PI3Kδ. Fur-
thermore, the model of the covalently bound amide ad-
duct that would form from these inhibitors did not reveal
any obvious conformational issues (Figure S1).
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Table 1. Compounds 2-9 inhibit PI3Kδ in biochemical
and cellular assays.
a
pIC50
hWB IFNγ
pIC50
b
δ
α
β
γ
Cpd
1
9.1
6.3
6.2
6.3
8.5
2
3
9.2c
8.3
8.1
8.2 c
5.6
5.5
5.6
4.8
5.1
7.2 c
5.1
5.8 c
4.6
4.8
4.9
5.0
N.T
8.1
4
5
6
7
8
9
5.3
7.9
7.9
7.4
6.9
7.0
5.0
6.7
Figure 1. Generalised design principles for development of
selective, irreversible PI3Kδ inhibitors.
8.2
7.3
6.4
7.4
7.9
8.3
5.4
4.9
4.8
<4.5
4.7
8.0
Activated esters potently engage PI3Kδ in enzyme
and cellular assays. A selection of phenolic esters were
synthesised, and their potencies at PI3Kα, β, γ and δ were
assessed using purified recombinant proteins via homo-
geneous time-resolved fluorescence (HTRF) assays.23 Ad-
ditionally, these compounds were tested in a phenotypic
human-whole blood (hWB) assay, measuring reduction in
interferon gamma (IFNγ) secretion after treatment with
T-cell stimulating antibody, CytoStim, as a readout for
PI3Kδ engagement23 (Table 1).
5.0
5.0
4.9
8.1
<4.5
4.8
8.2
10
Wortmannin
11
8.1
5.0
5.8
6.6
6.7
Zydelig
aBiochemical pIC50 data for all inhibitors at all four PI3K
isoforms (measured after 1 h at KM(ATP) using HTRF assays).
bPhenotypic hWB pIC50 derived from measuring levels of
IFNγ after stimulation with CytoStim (20 h incubation, free
compound concentrations are not available). Data for pan-
covalent PI3K inhibitor wortmannin 10, and FDA approved
PI3Kδ selective, reversible inhibitor Zydelig 11 derived from
these assays are also shown. cCompound found to be particu-
larly unstable in DMSO, results reported from N = 2 only.
N.T: Compound not tested due to instability of the DMSO 10
mM stock solution. All compounds were tested a minimum
of three times in HTRF and hWB assays, with the exception
of compounds 2, 8 and 9 (Table S1).
Esters 2-7 potently inhibited PI3Kδ in isolated enzyme
assays with pIC50 values ranging from micromolar (ester
7), to sub-nanomolar (ester 2), confirming that the phe-
nolic ester motifs were tolerated in the ATP binding
pocket of the kinase. Furthermore, these data suggested a
general trend that PI3Kδ potency of the inhibitors im-
proved with the electron withdrawing ability of the R
group attached to the ester, although the very high PI3Kδ
activity of 2 was achieved at the expense of selectivity.
The 4-trifluoromethylphenol ester 3 is an exception to
this trend. Its relatively strong electron withdrawing ef-
fect (σp = 0.78, 0.54, 0.06 and -0.27 for NO2, CF3, F, and
OMe substituents)43 did not enhance the PI3Kδ potency
compared to the electron neutral phenol 5. The decreased
potency of 2,4-dimethylphenol ester 7 may have arisen
from clash between the 2-methyl group and the kinase, or
steric hindrance to nucleophilic attack at the carbonyl.
Compounds 3 to 5 showed the best profiles in this analy-
Protein mass spectrometry and reactivity assess-
ments indicated the potential for site-specific nucle-
ophilic trapping by a lysine residue. After 5 min incu-
bation of recombinant PI3Kδ with 4 (2:1 molar ratio of
4:PI3Kδ), we observed formation of a single adduct by
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intact protein liquid chromatography-mass spectrometry
ences spanned two orders of magnitude, from 40 nM
(compound 2) to 7.8 µM (compound 6) (195-fold differ-
ence, Figure 2f). This indicated that the electronic nature
of the phenolate leaving group (i.e. pKa value), and there-
fore expected chemical reactivity/leaving group ability,
does not correlate with the rate of the covalent inactiva-
tion in this system. Rather, there is a correlation with KI,
suggesting a more complex mechanism than the tradi-
tional two-step scheme depicted in Figure 2e. The differ-
ences in pIC50 between the esters in our biochemical assay
must therefore be dictated by reversible interactions in
formation of the initial enzyme-inhibitor complex and
not the rate of the covalent reaction. We have proposed a
reaction mechanism supporting these data, invoking ad-
ditional steps to explain the dependence of KI on pKa
(Supplementary Discussion and Scheme S1). Finally,
our prior analysis of the chemical reactivity of 4 (Figures
S2 and S3) showed it to be inert to nucleophilic substitu-
tion under physiological conditions. This suggested that
the elevated reactivity observed in these kinetic analyses
was occurring specifically in the protein, and that the lo-
cal microenvironment around Lys779 may be contrib-
uting to an increased reactivity of this residue, rendering
it hyper-reactive.16,37,50,51
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3
4
5
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7
8
(LCMS). Compared to untreated protein, this mass shift
was consistent with the addition of 4, and loss of 4-
fluorophenol. Repeating this assay with 10 molar equiva-
lents of 4, and 20 h incubation showed no additional ad-
duct formation. Pre-incubation of PI3Kδ with 10 equiva-
lents of the potent ATP-competitive reversible inhibitor23
12 prevented formation of the covalent adduct, suggesting
covalent modification occurred in the ATP binding site.
Carboxylic acid 9 showed no evidence of covalent bond
formation in this experiment, implying that the phenolic
ester was required for the covalent reaction. These results
suggested that 4 was covalently, and specifically, binding
to the ATP binding site of PI3Kδ (Figure 2a).
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Reactivity analysis showed that 4 was stable to hydroly-
sis and reaction with N-Boc lysine under physiological
conditions (phosphate buffer, pH 7.4, 37 °C), but upon
deprotonation of the lysine smooth amide bond for-
mation was observed (Figures S2 and S3). Together with
the mass spectrometry data suggesting that a single, spe-
cific modification occurred in the active site of PI3Kδ, we
proposed that this bond could be forming with the con-
served lysine.
Confirmation of Lys779 as the nucleophilic residue.
After overnight soaking of pre-grown murine PI3Kδ crys-
tals20,23 with 4, we observed a covalently bound adduct
between the inhibitor and the targeted lysine residue
(Figure 2b) by X-ray crystallography. Continuous elec-
tron density was seen between Lys779 and the carbonyl of
the ester, and there was no evidence for the phenolic
group being present, consistent with formation of an am-
ide bond. Methyl ester 8 showed a reversibly bound ad-
duct (Figure S4), consistent with the reduced reactivity
of this ester (Figure S2). The remainder of the compound
satisfied the desired hydrogen bonding interactions be-
tween the indazole and hinge residues Val828 and
Glu826, as well as occupying the selectivity region next to
Trp760 with the basic amine.23,44
Selectivity, despite the conserved lysine, is driven
by reversible interactions. To assess off-target covalent
binding, jump dilution and kinetic measurements at two
closely related PI3K isoforms (α and β) were carried out
with 4. All three enzymes were inactivated in the jump
dilution experiment without competing ATP indicating
that 4 could covalently inhibit PI3Kα, β and δ at high pre-
incubation concentrations (Table S3), without a compet-
ing ligand (Figure 3a). As controls, the experiment was
performed with covalent pan-PI3K inhibitor 10, and re-
versibly bound ester analogues 8 and 9. 10 showed cova-
lent inactivation of all three kinases, whereas 8 and 9
showed the expected regeneration of enzyme activity af-
ter dilution, consistent with a reversible mode of inhibi-
tion (Figure 3a and Table S4).
Time-course experiments to determine kinact and
KI. Using the commercially available ADP QuestTM assay
kit45 we derived the concentration of inhibitor required
for half of the maximum rate of covalent bond formation
However, in the kinetic analysis with competing ATP (1
mM), linear reaction progressions were observed at PI3Kα
and β at all tested concentrations (Table S6). The gradi-
ent of these plots decreased with increasing concentration
of inhibitor, consistent with a rapid onset of inhibition.
The absence of the slow-binding non-linear phase we had
observed at PI3Kδ suggested no covalent binding was oc-
curring at these kinases under these conditions. Indeed,
for ester 4, the kinetics at PI3Kδ reflected 10, whilst the
kinetics at PI3Kα and β reflected the reversibly bound
methyl ester 8 (Figure 3b). This absence of apparent co-
valent adduct formation at PI3Kα and β was seen up to 8.5
μM of 4, ~5x the PI3Kδ IC99 (= 1.59 μM) under these con-
ditions. Reprocessing the raw data along the time axis
allowed derivation of IC50 values every 30 s over this time
window. These plots showed a time-dependent decrease
in IC50 at PI3Kδ, but not at PI3Kα and β for 4 (Figure S8),
supporting selective covalent inhibition in this assay. 4
also exhibited excellent selectivity against a panel of 10
lipid kinases and 140 protein kinases (Tables S7 and S8).
(KI), the rate constant for irreversible inactivation (kinact
)
and the second order rate constant typically used to char-
acterise irreversible inhibitors (kinact/KI) (Figure 2f).4,46–48
Full analyses for esters 2-7 and 10, including equations
used, are detailed in the Supporting Information.
All six esters exhibited non-linear reaction progress
curves (Figure 2c and Figure S7) indicating time-
dependent inhibition of PI3Kδ, consistent with covalent
inactivation. The kinact/KI ranking obtained from the
replot method correlated well with the potencies ob-
tained from the PI3Kδ biochemical and hWB assays
(Table 1 and Figure S7). The kinetic data were visualised
by plotting kinact as a function of KI, as described by
Schwartz et al. (Figure 2e).49 This representation clearly
showed that similar kinact values were found for all six es-
ters (1.4-fold difference across the series), but KI differ-
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Figure 2. Compounds 2-7 covalently inactivate PI3Kδ by amide bond formation to Lys779, with potency dependent on reversible
recognition. (a) Protein mass spectrometry. Top to bottom – apo protein; protein treated with two equivalents of 4, analysed at 5
min; protein treated with 10 equivalents of 4 overnight; protein pre-treated with 10 equivalents of 12 for 15 min, prior to addition
of two equivalents of 4, and analysed at 5 min; protein incubated with 10 equivalents of 9 and analysed at 5 min. (b) Left: Crystal
structure of 4 after overnight soaking with PI3Kδ crystals (PDB: 6EYZ). Right: Fo-Fc omit map is shown in green at 2.7 rmsd, with
Lys799 side-chain and ligand co-ordinates removed, showing clear electron density from Lys779 onto the ligand. (c) Raw time-
course data for 4. Concentrations are shown after correction for absolute stock concentration using quantitative NMR (Table
S5). (d) Plot of derived kobs from (b) vs concentration of inhibitor to derive KI and kinact. (e) Plot of derived kinact vs KI for all 6
esters, and wortmannin.4,46 (f) Table of kinact, KI and kinact/KI values. Kinetic measurements were performed in triplicate, and data
are
shown
as
the
mean
±
s.e.m.
These kinetic and jump dilution analyses revealed a
concentration window where 4 covalently inactivated
PI3Kδ, but not highly homologous isoforms of the same
family, despite the conserved nature of the nucleophilic
amino acid. Upon saturating the ATP binding site with
high concentrations of inhibitor, in the absence of com-
peting ATP, covalent inactivation does occur. However, in
a cell-like environment, covalent inactivation of PI3Kα
and β is unlikely to occur up to 5x the PI3Kδ IC99. Con-
sistent
with
our
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Figure 3. 4 does not covalently inhibit PI3Kα and β up to 5x PI3Kδ IC99 with cellular concentrations of ATP. (a) Jump dilutions
conducted at PI3Kα (left), PI3Kβ (middle) and PI3Kδ (right) in the absence of competing ATP. Covalent inactivation of all three
kinases was observed under these conditions by 4. Inhibitor assays were conducted in triplicate and controls in duplicate. Re-
sults are plotted as mean ± s.e.m (b) Kinetic plots of the inhibition over time under saturating ATP conditions (1 mM). Linear
plots (blue) relative to no-inhibitor controls (black) are typical of reversible inhibitors. Non-linear plots (red) relative to no-
inhibitor controls (black) are typical of slow-binding inhibitors (confirmed to be irreversible by jump dilution experiments in
(a)). The top row depicts reversible ester 8, showing linear progress at IC90 at PI3Kδ, and at PI3Kα and β at the PI3Kδ IC99. Mid-
dle row depicts irreversible inactivation of all three kinases by wortmannin at IC90 for PI3Kδ (1.5x PI3Kδ IC90 curves are also
shown). Bottom row shows selective ester 4, exhibiting covalent inhibition at PI3Kδ at IC90, and reversible inhibition at PI3Kα
and β at 3x and 5x PI3Kδ IC99. Assays were conducted in duplicate, and results are shown as mean ± s.e.m.
earlier observation that potency differences between the
esters at PI3Kδ was governed by reversible interactions,
this selectivity must also arise from differences in the ini-
tial reversible binding interactions (KI) with these kinases.
strain-promoted azide-alkyne cycloaddition click reaction
(SPAAC) with dibenzycyclooctyne-conjugated-Cy5 dye
(DBCO-Cy5), and SDS-PAGE separation. Co-elution of
PI3Kδ with the Cy5 signal was confirmed by im-
munostaining with p110δ antibody after transfer to PVDF
membranes (Figure S9).52 Furthermore, the Cy5 fluores-
cence signal was gradually ablated in the presence of in-
creasing concentrations of 4, with a pIC50 of 7.5, suggest-
ing potent engagement of PI3Kδ by 4 in complex cell ly-
sates.
Chemoproteomics revealed 4 to be selective for
PI3Kδ in live cells. Azido probe 13 was synthesised, and
retained potency at PI3Kδ in the biochemical HTRF assay
(pIC50 = 7.6). At 1 μM, it was shown to covalently modify a
protein with a molecular weight consistent with PI3Kδ in
THP-1 monocyte lysates by in-gel fluorescence after
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Figure 4. Chemoproteomic analysis identifies targets of 4 in human cells. (a) General assay design. (b) Structures of 4 and 13,
and key for graphs below. (c) The scatter plots display proteins enriched with neutravidin beads after clicking a biotin moiety to
proteins labeled in situ with probe 13 (1 μM) relative to vehicle control. Proteins were accessed by enzymatic bead digestion or
SDS elution from the bead. The dotted lines represent 2-fold enrichment (13/DMSO) and proteins with >2-fold enrichment are
deemed specific targets of 13, and are labeled by their entrez gene ID. (d) Dose-response curves for competition of 13 binding to
specifically enriched targets after pretreatment of cells for 1 h with 4. Binding curves and resulting pIC50 values are shown for
proteins enriched >2-fold. With the exception of FECH, values are not plotted if no binding curve could be fitted using
GraphPad Prism 7.03. The values represented are the average ± s.e.m. from two biological replicates. In (c) and (d), data were
filtered with the criteria that quantified unique peptide matches > 1 and quantified unique peptide to spectra matches >2. Pro-
teins were required to be identified and quantified in both replicates to be included in the analysis, and data are plotted as the
mean of the TMT label ratios (MS2) for the three most abundant peptides (MS1) per protein.
Protein targets of probe 13 in Ramos cells were then
identified by a quantitative mass spectrometry-based
chemoproteomics method using tandem mass tag (TMT)
labelling (Figure 4a), based on related experiments by
Lanning et al53 and Niessen et al54. Cells were treated with
either vehicle or 13 at 1 μM for 1 h, lysed, and proteins
bound by 13 were enriched with neutravidin beads after
SPAAC reaction with a DBCO-biotin conjugate. Non-
covalently bound proteins were expected to be predomi-
nantly eluted with SDS, whereas covalent targets should
only be detected after on-bead proteolysis.
protein RUBCNL-like (gene ID C13ORF18) as well as the
common kinase inhibitor off-target ferrochelatase56 (gene
ID FECH), were only found in the SDS eluates. The possi-
bility to elute those proteins from the capturing matrix
with SDS buffer implicates those proteins as reversible
binders of 13, or proteins in complexes with enriched tar-
gets. The PI3K regulatory subunit α (gene ID PIK3R1) was
identified in the bead digest fraction as well as in the SDS
eluates. Signal abundances of detected tryptic peptides
(MS1 intensities) indicated a 3 to 4-fold higher abundance
in the SDS fraction than in the bead digests. This suggests
major, but incomplete, elution of this known interactor of
class I catalytic PI3K subunits26 with the applied condi-
tions.
Comparison to vehicle-treated cells identified 22 out of
~1000 identified proteins that were specifically enriched
>2-fold by in situ treatment with 13 (Figure 4c and Sup-
plementary Data Set). Of those 22 proteins, eight were
exclusively identified after direct proteolysis, including
the class I catalytic subunits of PI3Kδ (gene ID PIK3CD),
PI3Kα (gene ID PIK3CA), PI3Kβ (gene ID PIK3CB), and
the class III PI3K protein Vps34 (gene ID PIK3C3), sug-
gesting these proteins as covalent targets of 13. In con-
trast, the Vps34 regulatory subunits55 PI3K regulatory
subunit 4 (gene ID PIK3R4), Beclin-1 (gene ID BECN1),
p63 (gene ID UVRAG), mitofilin (gene ID IMMT), and
To accurately assess off-target interactions of 4, we de-
rived dose-response curves from competing the binding
to 13 by pretreatment with concentrations of 4 ranging
from 10 µM to 3.2 nM for 1 h (Figure 4d). For the specifi-
cally enriched proteins exclusively found in the bead di-
gests, we calculated pIC50 values for PI3Kδ, PI3Kα, PI3Kβ,
and hVps34 to be > 8.5, 7.0, 6.9 and 7.2, respectively. This
indicates that irreversible binding of PI3Kδ to 13 can be
competed by 4 with >30-fold selectivity at the used incu-
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results are depicted as mean ± s.e.m. Non-washed curves
showing the dose-response after 48 h are shown in blue.
bation conditions. For the proteins identified exclusively
in the SDS fraction, we determined reasonable binding
curves for Beclin-1, p63, and PI3K regulatory subunit 4. All
resulted in very similar pIC50 values (pIC50,BECN1 = 6.8;
pIC50,UVRAG = 6.6; pIC50,PIK3R4 = 6.6). Binding curves were
incomplete for those proteins (maximal competition by
10 µM compound c.a. 50%), suggesting they were interact-
ing with a known complex partner,55 rather than being
true targets of the compound. A similar effect might be
observed for PI3K regulatory subunit α for which a pIC50
value >8.5 was determined. As a complex partner of
PI3Kα, PI3Kβ, and PI3Kδ,26 the determined apparent dose-
dependent competition may result from a combination of
competitive binding to any of these PI3K proteins. Within
the tested concentration range, mitofilin, ferrochelatase,
and protein RUBCNL-like, (as well as the remaining pro-
teins that were specifically enriched with 13) did not show
strong competition of binding by 4, suggesting low affini-
ty binding.
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CONCLUSION
Selective covalent inhibition relies on a two step pro-
cess of reversible binding and then covalent
inactivation.4,5 Here, we have demonstrated that selectivi-
ty in formation of the initial enzyme-inhibitor complex is
the crucial factor for achieving potent, selective covalent
inhibition of conserved residues. Kinetic studies indicated
that the electronics, and expected chemical reactivities, of
the electrophilic esters did not affect the rate of covalent
bond formation with the enzyme, kinact. The observed var-
iation in potency of the esters at PI3Kδ (Table 1) therefore
arose from the reversible binding steps of the inhibitors,
due to electronic changes in the phenolic group, reflected
by KI. Furthermore, through kinetic studies at PI3Kα, β,
and δ we determined that there was a reasonable concen-
tration range at which covalent inhibition of PI3Kδ could
be achieved, without covalent inactivation of related en-
zymes. This was confirmed by chemoproteomic studies
with 13 in situ which suggested minimal off-target binding
of 4, and a >30-fold selectivity window for covalent modi-
fication of PI3Kδ, despite conservation of the targeted
lysine residue and presence of other reactive moieties.
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These chemoproteomic data support minimal off-target
binding by 4 in human cells. Coupled with our selectivity
hypothesis and kinase panel investigations above (Figure
3 and Tables S7 and S8), we propose 4 to be a highly se-
lective irreversible inhibitor of PI3Kδ at concentrations
below 1 μM.
Cellular washout studies suggest an extended du-
ration of action. CD4+ T cells were incubated with 4 or
12 (hWB pIC50 = 7.9) for 2 h, then washed and incubated
for a further 48 h before stimulation with αCD3 and
measurement of IFNγ release. 4 showed sustained deple-
tion of IFNγ secretion 48 h after washout, without cyto-
toxic effects (Figure S10), whereas regeneration of IFNγ
secretion was observed for 12 (Figure 5). These results,
taken together with the data presented above, suggest
that inhibition of PI3Kδ could be sustained for at least 48
h using an irreversible targeting approach, with no cyto-
toxic effect. It is worth noting, however, that cellular ac-
cumulation of dibasic piperazines could also be a contrib-
uting factor to the duration of action for this series of
compounds.57,58
As a more general approach, variation of the electro-
philic centre to affect reversible binding could be exploit-
ed for fine-tuning of the potency, selectivity, and physico-
chemical properties of inhibitors for irreversible drug dis-
covery programmes. By maintaining a constant kinact
across a series of electrophilic esters, this provides an or-
thogonal approach to established EGFR and Janus Kinase
(JAK) inhibitors that vary the kinact to improve the drug
profile.8,59 Furthermore, a common method for enzymes
to develop resistance to covalent inhibitors targeting
poorly conserved cysteines is by point mutation of the
modified residue.60 A similar resistance mechanism would
not be applicable to the strategy we have developed, as
mutation of the catalytic lysine would render the kinase
inactive.61 Finally, the potential lability of these esters
could impart kinetic selectivity in vivo. Zaro et al. recently
described how the proteome-wide selectivity of ibrutinib
improved when a fumarate ester was incorporated into
the covalent warhead. They attributed this to hydrolysis
of the metabolically labile ester, affording an inert 1,4-
unsaturated carboxylate.62 Carboxylic acid 9 formed in
our case is poorly active in the cellular assay (pIC50 = 5.0,
Table 1), and inert to covalent bond formation by mass-
spectrometry (Figure 2a), suggesting a similar mecha-
nism could be observed here.
Figure 5. Inhibitor 4 showed a prolonged duration of action,
up to at least 48 h in CD4+ T cells. Cellular washout studies
conducted in CD4+ T cells isolated from hWB. Cells were
treated with inhibitors for 2 h, washed, and stimulated with
αCD3 to induce IFNγ release, measured after 48 h. Left: Co-
valent compound 4 showed a clear sustained duration of
action (black circles) 48 h after washing. Right: Reversible
compound 12 showed a clear disruption of the inhibition
profile (black circles) 48 h after washing. The experiment was
repeated using 5 donors (for each donor: N = 3 replicates for
washout, and N = 2 replicates for non-wash condition), and
By modification of a known reversible inhibitor,23 we
have developed a series of esters which selectively and
covalently inhibit PI3Kδ. In addition to the excellent se-
lectivity profile, our lead compound 4 showed ~10 nM
activity in the hWB phenotypic inflammatory cytokine
response assay, and extended duration of action (>48 h),
in cellular washout studies. Owing to the importance of
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PI3Kδ as a target for inflammation and oncology, this se-
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lective, covalent PI3Kδ inhibitor could have applications
in the development of long-acting therapeutics.
Prior studies have shown that the targeted conserved
lysine reacts covalently with promiscuous probes pos-
sessing reactive warheads.38–41 We therefore envisage that
this approach could be orthogonal to non-conserved cys-
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method, researchers may be able to generate selective
covalent chemical probes of any chosen kinase, which
could provide tools to vastly improve the understanding
of human biology in diseased states.
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ASSOCIATED CONTENT
Supporting Information.
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Biological methods, synthetic methods and characterisation,
supplementary figures, supplementary tables, supplementary
mass-spectrometry tables, and supplementary discussions.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
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* sebastien.x.campos@gsk.com
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interests.
(24)
ACKNOWLEDGMENT
We thank the GlaxoSmithKline/University of Strathclyde
Industrial PhD programme for funding this work. We also
thank D. House for providing revisions of the manuscript,
J.N. Hamblin and V. Patel for intellectual discussions, and N.
Barton, C. Chitty, P. Francis, L. Gordon, N. Hodnett, S.
Kumper, S.M. Lynn, J.E. Rowedder, E. Sherriff and H. Taylor
for technical assistance and discussion.
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