Discovery of a potent and isoform-selective targeted covalent inhibitor of the lipid kinase PI3Kα

Article abstract of DOI:10.1021/jm3008745

PI3Kα has been identified as an oncogene in human tumors. By use of rational drug design, a targeted covalent inhibitor 3 (CNX-1351) was created that potently and specifically inhibits PI3Kα. We demonstrate, using mass spectrometry and X-ray crystallography, that the selective inhibitor covalently modifies PI3Kα on cysteine 862 (C862), an amino acid unique to the α isoform, and that PI3Kβ, -γ, and -δ are not covalently modified. 3 is able to potently (EC₅₀ < 100 nM) and specifically inhibit signaling in PI3Kα-dependent cancer cell lines, and this leads to a potent antiproliferative effect (GI₅₀ < 100 nM). A covalent probe, 8 (CNX-1220), which selectively bonds to PI3Kα, was used to investigate the duration of occupancy of 3 with PI3Kα in vivo. This is the first report of a PI3Kα-selective inhibitor, and these data demonstrate the biological impact of selectively targeting PI3Kα.

Full text of DOI:10.1021/jm3008745

Article
pubs.acs.org/jmc
Discovery of a Potent and Isoform-Selective Targeted Covalent
Inhibitor of the Lipid Kinase PI3Kα
Mariana Nacht,* Lixin Qiao, Michael P. Sheets, Thia St. Martin, Matthew Labenski, Hormoz Mazdiyasni,
Russell Karp, Zhendong Zhu, Prasoon Chaturvedi, Deepa Bhavsar, Deqiang Niu, William Westlin,
Russell C. Petter, Aravind Prasad Medikonda, and Juswinder Singh
Celgene Avilomics Research, 45 Wiggins Avenue, Bedford, Massachusetts 01730, United States
S
* Supporting Information
ABSTRACT: PI3Kα has been identified as an oncogene in human
tumors. By use of rational drug design, a targeted covalent inhibitor 3
(CNX-1351) was created that potently and specifically inhibits PI3Kα.
We demonstrate, using mass spectrometry and X-ray crystallography,
that the selective inhibitor covalently modifies PI3Kα on cysteine 862
(C862), an amino acid unique to the α isoform, and that PI3Kβ, -γ,
and -δ are not covalently modified. 3 is able to potently (EC50 < 100
nM) and specifically inhibit signaling in PI3Kα-dependent cancer cell
lines, and this leads to a potent antiproliferative effect (GI50 < 100
nM). A covalent probe, 8 (CNX-1220), which selectively bonds to
PI3Kα, was used to investigate the duration of occupancy of 3 with
PI3Kα in vivo. This is the first report of a PI3Kα-selective inhibitor,
and these data demonstrate the biological impact of selectively
targeting PI3Kα.
inhibitor that does not bind to β, δ, and γ may spare toxicities
caused by prevention of platelet aggregation and activation,13
inhibition of immune responses mediated by T, B, and mast
cells,14 and blocking anti-inflammatory signals,15 respectively.
Recently, there has been a resurgence of interest in the use of
covalent inhibitors to address challenges in potency and
selectivity to drug targets.16 There are many examples of
covalent drugs that have proven to be safe and successful
therapies for a wide variety of indications, including blockbuster
drugs such as esomeprazole (AstraZeneca) and clopidogrel
(Sanofi-Aventis/Bristol-Myers Squibb).16 The majority of these
covalent drugs have been discovered serendipitously, but
recently there has been progress in the rational design of
covalent drugs, termed targeted covalent inhibitors, which has
been described mainly in the area of kinases17 and more
recently in the protease field.18 Here we describe the first
example of a targeted covalent inhibitor of the lipid kinase
family that is an isoform-selective inhibitor of PI3Kα.
INTRODUCTION
■
The family of lipid phosphoinositide 3-kinases (PI3Ks) plays a
key regulatory role in many cellular processes including
proliferation, cell survival, differentiation, and metabolism
(reviewed in refs 1 and 2). There are three classes of PI3Ks.
The class I family is the best studied and the family most
implicated in cancer cell growth. The class I PI3Ks are
heterodimers consisting of a p110 catalytic subunit and a p85
regulatory subunit. The PI3K class I family consists of four
isoforms, α, β, γ, and δ. PI3Ks α and β are ubiquitously
expressed, whereas expression of γ and δ is more restricted,
mostly to leukocytes. The lipid kinases convert phosphatidy-
linositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-tri-
sphosphate, and the process is reversed by the phosphatase
and tumor suppressor protein PTEN.3,4 In recent years,
activated class IA PI3K signaling has been increasingly linked
to cancer. PI3K can be activated by receptor tyrosine kinase
(RTK) signaling1,5−7 and inactivation of the negative regulator
PTEN,8 and through direct somatic mutations in PIK3CA, the
gene that encodes the p110α subunit of PI3K.1,9 PIK3CA is
mutated in up to 30% of human cancers, including breast,
colon, prostate, and endometrial cancers.1,10 Importantly, it has
been demonstrated that these mutations are in fact
oncogenic.11
DESIGN OF A TARGETED COVALENT INHIBITOR
OF PI3Kα
■
A major challenge in the design of isoform-selective PI3K
inhibitors has been the high similarity of the lipid kinase ATP
binding sites. We examined the ATP binding site and nearby
residues of PI3Kα to identify opportunities for selective
Several PI3K inhibitors are currently in clinical development,
but most are pan-PI3K inhibitors that bind reversibly to the
ATP site.12 The human tumor genetic data suggest that
mutations in PI3Kα drive tumor growth and therefore support
the need for a PI3Kα selective inhibitor. Moreover, a selective
Received: June 21, 2012
Published: January 29, 2013
© 2013 American Chemical Society
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Figure 1. Binding site sequence and structure of PI3Kα complexed with compound 3. (a) Binding site composition of class 1 PI3K isoforms α, β, γ,
and δ. (b) X-ray complex of 3 to PI3Kα. The protein backbone is shown as a cartoon. The structure of 3 is shown as a stick representation, and the
binding site residues are shown as lines. Hydrogen bonds between 3 and the protein are shown as dashed lines. C862 is highlighted, as well as D810,
Y836, and V851 (hinge region) which are involved in hydrogen bonds with the inhibitor. (c) Overlay of 3−PI3Kα (yellow) X-ray complex with X-
ray complex of GDC-0941 bound to PI3Kγ (3DBS, Ggreen). (d) Electron density of 3 bound to PI3Kα. The ligand and neighboring protein side
chains are shown as a stick model, colored according to the chemical atom type (3 in cyan, human PI3K in gray, N in blue, O in red, and S in
yellow). The ligand molecule is shown superimposed with the refined 2Fo − Fc electron density map contoured at 1.0.
covalent modification. Our design strategy was to use the
binding mode of the reversible pan-PI3K inhibitor GDC-
094119 to create a targeted covalent inhibitor. The binding
mode of GDC-0941 to PI3Kγ had been determined by X-ray
crystallography, and we used molecular modeling to model the
binding of GDC-0941 to the X-ray structure of PI3Kα.20 By
aligning the PI3Kα model with the X-ray complex of PI3Kγ, we
identified C862 as a promising amino acid to target, since it is
near the small molecule binding site (∼10 Å), provides a
relatively strong nucleophilic center, and is unique to PI3Kα (it
is not present in β, γ, and δ; Figure 1a). Through a series of
design cycles exploring both linker spacing and electrophilic
functional groups, we identified 3 (CNX-1351) as a targeted
covalent inhibitor of PI3Kα (Figure 2a). In addition, we
developed an irreversible biotinylated covalent probe 8 (CNX-
1220; see Figure S1 in Supporting Information and Scheme 2)
that allows for the measurement of free protein versus bound
target protein.
Figure 2. 2D chemical structure of (a) compound 3 and (b) GDC-
0941.
coupling with (1H-indazol-4-yl)boronic acid to give 4-
indazolylthienopyrimidine 2. After Boc deprotection, the
resulting piperazine was acylated with carbonyldiimidazole-
activated 6-methyl-4-oxohept-5-enoic acid, which delivered the
desired gem-dimethylenone compound 3.
The biotinylated covalent probe 8, was designed to measure
the occupancy of PI3Kα by covalent inhibitors (i.e., 3). The
synthesis of 8 is described in Scheme 2. Compound 1 was
treated with 3-hydroxyphenylboronic acid under Suzuki
conditions to give phenol 5. Upon deprotection, the resulting
piperazine was treated with 5-(diethoxyphosphoryl)-4-oxopen-
CHEMISTRY
■
The synthetic approach for the covalent inhibitor 3 (Figure 2a)
is outlined in Scheme 1. Thienopyrimidyl chloride 1 was
prepared using methods similar to those reported for GDC-
0941 (Figure 2b).21,22 Compound 1 then underwent Suzuki
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a
Scheme 1
a
Reagent and conditions: (a) (1H-indazol-4-yl)boronic acid, Na2CO3, H2O, Pd(PPh3)2Cl2, DMA, microwave, 120 °C, 1 h, 73%; (b) HCl in dioxane,
DCM−MeOH, 100%; (c) 6-methyl-4-oxohept-5-enoic acid, carbonyl diimidazole, DMA, rt, 45 min; then de-Boc intermediate, DIPEA, DMA, rt,
overnight, 62%.
a
Scheme 2
a
Reagent and conditions: (a) 3-hydroxyphenylboronic acid, Pd(PPh3)4, Na2CO3, toluene/ethanol/H2O, 120 °C, 1 h, 69%; (b) HCl in dioxane,
DCM−MeOH, 100%; (c) compound 3, carbonyldiimidazole, DMA, rt, 45 min; then de-Boc intermediate, DIPEA, DMA, rt, overnight, 47%; (d)
K2CO3, DMA−H2O, 70 °C, 4 h; (e) CF3CO2H, dichloromethane; (f) N-biotinyl-NH-(PEG)2-COOH-DIPEA (20 atoms), HATU, DIPEA, DMA.
tanoic acid 4, which was prepared by condensing succinic
anhydride with deprotonated diethyl methylphosphonate. The
resulting phosphonate 6 subsequently underwent Wittig−
Horner−Emmons reaction with tert-butyl 4-formylbenzylcarba-
mate, giving trans-enone 7 only. Final assembly of covalent
probe 8 was achieved via de-Boc of compound 7 followed by
amide formation with commercially available polyethylene
glycol linked biotin glutaric acid.
RESULTS AND DISCUSSION
■
Mass spectrometry was used to confirm that 3 covalently bonds
to PI3Kα but not to the other class I isoforms (see Figure S2a
in Supporting Information). To confirm that C862 was the
amino acid on which the bond was formed, PI3Kα protein was
incubated with 3 and then subjected to tryptic digestion.
Subsequent peptide analysis of the digested, modified protein
confirmed modification of C862 (see Figure S2b in Supporting
Information).
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Using X-ray crystallography, we confirmed our predicted
binding mode of 3 to PI3Kα (Figure 1b). The X-ray complex of
GDC-0941 has been described previously, binding to PI3Kβ, -γ,
and δ, and has been shown to adopt a three-point binding
mode to the PI3K ATP site.19 Comparison of the published
binding mode of GDC-0941 (PDB code 3DBS) with our X-ray
complex of 3 to PI3Kα shows similar binding modes in the
PI3K ATP binding site but also important differences (Figure
1c). The two compounds adopt a similar packing arrangement
of the thienopyrimidine core. Both employ the morpholine
oxygen to form a hydrogen bond with the hinge region and
place the indazole into the affinity pocket. There are differences
in the binding mode of the substituted piperazine functionality
between the two compounds. Unlike the binding mode of
GDC-0941, in which the piperazine is kinked out of the plane
relative to the thienopyrimidine core and packs against Arg770
and Ser773, the substituted piperazine of 3 is directed toward
the other side of the binding site toward the side chain of C862.
The dimethylenone is clearly seen in the electron density to
form a covalent bond with C862 (Figure 1d).
dimethylenone of 3 would react with the thiol present in GSH.
After 2 h at 37 °C, >80% of free 3 was recovered, consistent
with the no-GSH control samples (see Figure S3a in
Supporting Information). This indicates that 3 is not
indiscriminately reactive toward GSH.
3 was incubated in albumin-depleted human plasma at 37 °C
for 1 or 3 h to determine whether it would covalently bond
with any highly abundant human plasma proteins. None of the
observed human plasma proteins showed mass shifts consistent
with the mass of 3, suggesting an inability to covalently modify
these off-target plasma proteins at detectable levels (see Figure
S3b in Supporting Information).
Compound 3 Inhibits PI3Kα Signaling in Cells and
Shows Prolonged Inhibition Consistent with a Covalent
Mechanism of Action. SKOV3 ovarian cancer cells have a
mutation in PIK3CA that results in a constitutively activated
PI3Kα signaling pathway.23 These cells were used to examine if
3 could inhibit P-AktSer473 as a measure of inhibition of PI3K
signaling. 3 (1−1000 nM) was added to SKOV3 cells for 1 h.
Lysates of the cells were interrogated by immunoblot analysis
for P-AktSer473. A noncovalent pan-PI3K inhibitor, GDC-0941,
was used as a control and demonstrates that 3 inhibits PI3K
signaling in SKOV3 cells, with potency (EC50 of 10−100 nM;
see Figure S4 in Supporting Information) similar to that of the
pan-PI3K inhibitor.
To demonstrate the covalent mechanism of action in cells,
SKOV3 cells were transiently treated with the covalent
inhibitor, and prolonged inhibition of P-AktSer473 was examined.
SKOV3 cells were incubated with 3 or the reversible inhibitor
GDC-0941 (500 nM each) for 1 h. Cells were washed, and
medium was replaced with fresh compound-free medium at
regular intervals (approximately once every hour for 3 h). Cell
lysates were prepared at 1, 6, and 24 h after compound removal.
Cell lysates were interrogated by immunoblot analysis for P-
AktSer473 (Figure 3). As expected, signaling returned immedi-
ately after removal of the reversible inhibitor, GDC-0941, but
was inhibited >6 h after removal of 3, consistent with the
activity of an irreversible compound.
Compound 3 Inhibits Growth of Cells Dependent on
PI3Kα. To investigate the functional consequence of inhibiting
PI3Kα in cells, two cell lines with different PIK3CA activating
mutations, SKOV3 ovarian cancer cells (H1047R) and MCF-7
breast cancer cells (E545K), were treated with 3 and growth
was monitored. Both PIK3CA-driven cell lines were growth
inhibited by exposure to 3 for 96 h (GI50 of 78 and 55 nM,
respectively) (Table 2). These data suggest that selective
inhibition of PI3Kα will lead to an antiproliferative effect on
PIK3CA-driven tumors.
Compound 3 Inhibits PI3Kα Signaling in Vivo and
Bonds to p110α. To examine the activity of 3 on PI3Kα
signaling in vivo, 3 was delivered to nude mice (100 mg/kg ip)
once a day for 5 consecutive days. Because PI3Kα is
ubiquitously expressed, spleens were harvested from the mice
at 1, 4, and 24 h after the last administration of compound to
measure the impact of 3 on PI3K signaling. Lysates from the
spleens were immunoblotted for P-AktSer473 (Figure 4a).
Inhibition of PI3K signaling was detected as a decrease in P-
AktSer473 at 1 and 4 h after the last dose.
Compound 3 Is a Selective Covalent Inhibitor of
PI3Kα. The biochemical potency and selectivity of 3 were
examined. 3 was tested against all four of the class I PI3K
enzymes α, β, γ, and δ. In an end point assay, 3 potently
inhibited PI3Kα and was 20−400 times less potent against β, γ,
and δ (Table 1). Because of the irreversible mechanism of
a
Table 1. Biochemical Selectivity of Compound 3
b
kinase
IC50_App
PI3Kα
PI3Kβ
6.8 nM
166.0 nM
240.3 nM
3020.0 nM
>1 μM
PI3Kγ
PI3Kδ
PI3KC2A
PI3KC3
PI4Kα
PI4Kβ
>1 μM
>1 μM
>1 μM
SPHK1
SPHK2
>1 μM
>1 μM
a
Compound 3 was tested in a panel of 10 lipid kinases, and an IC50
b
apparent was determined (IC50_App). Irreversible inhibitors show
time-dependent inhibition. Thus, an IC50 apparent is used to
determine the inhibitory activity of irreversible molecules at a given
time point.
action, 3 shows time-dependent inhibition. Thus, IC50
apparents are used to report the inhibitory activity of 3 at a
given time point. 3 showed even greater selectivity against the
class II lipid kinases, none of which were appreciably inhibited
at the highest concentration of drug tested (1 μM) (Table 1).
Moreover, 3 was tested against a panel of 60 kinases,
representing all branches of the kinome tree, and showed
excellent selectivity; no kinases were inhibited by greater than
40% using 1 μM compound (see Table S1 in Supporting
Information), suggesting that 3 is a very selective PI3Kα
inhibitor.
Compound 3 Shows No Reactivity toward Gluta-
thione or Abundant Human Plasma Proteins. The off-
target reactivity of 3 was assessed using mass spectrometry
methods. 3 was reacted with a 500-fold molar excess of
glutathione (GSH), a tripeptide and abundant intracellular
thiol, in a metabolism-free setting to determine whether the
An irreversible covalent inhibitor allows for the measurement
of free protein versus bound target protein using a covalent
probe that bonds only to free protein. A biotinylated covalent
probe that is selective for p110α (8; see Figure S5a and Figure
S5b in Supporting Information) was used to detect free,
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PI3Kα but not in the other isoforms has enabled us to achieve
selective covalent inhibition of PI3Kα. This is in contrast to the
natural product wortmannin, which is also a covalent inhibitor
of the PI3K family but which targets a conserved Lys and
therefore shows no isoform selectivity.
This is also the first reported X-ray structure of a targeted
covalent inhibitor to the lipid kinase family. Our predicted
binding model of 3 was in good agreement with the PI3Kα X-
ray crystal structure that we determined. We have recently
compared the reactivity and geometry of the β,β-dimethyle-
none that is present in 3 with other five α,β-unsaturated
ketones using high level quantum mechanical calculations.24
The β,β-dimethylenone showed the highest activation barrier in
aqueous solution, indicating that it is a highly stable electrophile
compared to α- or β-methylenone and α,β-dimethylenone. The
covalent mechanism of action enables complete and durable
inhibition of PI3Kα, which leads to silencing of the target
protein and its downstream oncogenic signaling. Our data show
that the inhibition of PI3Kα persists even after washout of drug,
which could enable less frequent dosing and minimize off-target
activity. Less frequent dosing coupled with specificity toward
the α isoform may enable a higher therapeutic index by sparing
potential toxicities from pan inhibition of this central lipid
kinase family with pleiotropic functions. Another important
benefit of the covalent strategy is the companion covalent
probe that was used preclinically to assess occupancy of PI3Kα
in vivo and could be a powerful translational tool in assessing
PK/PD in the clinic.
3 is a powerful tool compound that will allow several
important questions about PI3K biology to be addressed. The
selectivity for PI3Kα and the prolonged duration of inhibition
will allow the investigation of the role of PI3Kα in different
tumor models with different genetic contexts. Moreover, there
are reports regarding the contribution of α and β to glucose
homeostasis offering diverging conclusions; a selective α
inhibitor may resolve these open questions. Although 3 has
useful properties as a tool compound, it also has deficiencies as
a lead compound. The pharmacokinetic properties of the
compound are suboptimal and are the focus of a medicinal
chemistry effort to improve its oral bioavailability while
maintaining its potency and selectivity. This will be the focus
of a future publication. Finally, emerging clinical data support
the notion that combinations of targeted therapies may be
more effective than monotherapy and may help reduce the
emergence of drug resistance. Thus, a selective and durable
inhibitor of PI3Kα may be a very important component of
future combination approaches.
Figure 3. Inhibition of p-AktSer473 continues after removal of
compound 3. SKOV-3 cells were incubated with 3 or GDC-0941
(500 nM each) for 1 h. Medium was replaced with fresh compound-
free medium at regular intervals, and cell lysates were prepared at the
indicated times after compound removal. Cells were processed for in-
cell Western analysis (see Experimental Section) to determine
expression of P-AktSer473. Signaling returned immediately after removal
of the reversible inhibitor, GDC-0941, but was inhibited >6 h after
removal of 3. Data shown are from duplicates. Experiment was
performed at least 3 times: (∗∗) p < 0.001; (∗) p < 0.05.
Table 2. Compound 3 Inhibits Growth of Cells Dependent
a
on PI3Kα
GI50 SEM (nM)
SKOV3 (PIK3CAH1047R
)
MCF-7 (PIK3CAE545K
)
GDC-0941
147.6 36.6
77.6 16.8
70.2 13.5
54.7 14.3
3
a
Cells with activating mutations in PI3KCA (H1047R or E545K) were
growth inhibited by the PI3Kα selective compound 3 or the pan-PI3K
inhibitor GDC-0941. Tumor cells (3000−7000 cells/well) were placed
in a 96-well plate and allowed to settle for 5 h. Compounds were
serially diluted (1 μM to 4 nM) and added to the cells. After a 96 h
incubation, cell viability was measured using Cell Titer-Glo (Promega)
to determine the growth inhibition. Data shown are an average of four
experiments, each performed in duplicate.
uninhibited p110α in spleen cells. There was little free p110α at
1 and 4 h after the last dose, indicating that 3 irreversibly
bonded to p110α in vivo (Figure 4b). At 24 h after the final
dose, free p110α was detectable because of protein resynthesis,
concomitant with the return of P-AktSer473 expression.
EXPERIMENTAL SECTION
■
Chemistry. General Experimental Details. All commercial
reagents and anhydrous solvents were obtained from commercial
sources and were used without further purification unless otherwise
specified.
Microwave chemistry was done using CEM Explorer. LC−MS was
used to analyze the purity of all compounds on an Agilent LC-1200
series coupled with Agilent 6120 quadruple mass spectrometer. The
mobile phase for all mass analysis consisted of acetonitrile−water
mixtures with 5 mM ammonium acetate and 5 mM acetic acid. All final
compounds were greater than 95% pure. Preparative reverse-phase
chromatography was carried out using using a Prep Varian ProStar
model 210 on a Microsorb-MV 100-8 C18 Dynamax 250 mm × 21.4
mm column with a linear gradient from 10% to 90% CH3CN in H2O
over 20 min (0.1% trifluoroacetic acid) and a flow rate of 20 mL/min.
1H and 13C NMR spectra (δ, ppm) were recorded on a JEOL AMX-
CONCLUSION
■
This is the first detailed report of a selective covalent inhibitor
of PI3Kα. Recently, Novartis and Intellikine launched phase I
clinical trials for α-selective inhibitors (BYL719 and INK1117,
respectively, AACR 2012), but detailed reports of these drugs
have not been published. Our data demonstrate that the
application of a targeted covalent inhibitor strategy to PI3Kα
has a number of unique features related to the covalent
mechanism of action. The targeting of a Cys which is present in
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Figure 4. Compound 3 inhibited p-AktSer473 in mouse spleens and bonds to PI3Kα in vivo. Compound 3 was delivered into the intraperitoneal cavity
of nude mice at 100 mg/kg once a day for 5 consecutive days (n = 3 mice per group). Spleens were harvested from the mice at the indicated times
after the last dose (1−24 h) and interrogated by immunoblot for P-AktSer473 (a) or for PI3Kα occupancy (b) as described in Experimental Section.
Inhibition of PI3K signaling was detected as a decrease in P-AktSer473 at 1 and 4 h after last dose. Similarly, there was little/no free PI3Kα at 1 and 4 h
after the last dose ((∗∗) p < 0.001). Protein resynthesis accounts for the return of free protein and signaling activity at 24 h ((∗∗) p < 0.001).
400 (400 MHz) or Bruker AVANCE-3 (400 MHz) instrument. High-
resolution mass spectra were measured using a Waters Q-Tof Premier
operating in +ESI W-mode.
dimethylacetamide for 30 min before transferring into the de-Boc
intermediate above in 1 mL of N,N-dimethylacetamide. The reaction
mixture was stirred overnight, then subjected to normal EtOAc/H2O
workup. After solvent removal, the resulting solid was filtered and
washed with acetonitrile, giving 36 mg of the desired product as a pale
white solid (62%). 1H NMR (400 MHz, CDCl3): δ 8.97 (s, 1H), 8.22
(d, 1H, J = 6.4 Hz), 7.52 (d, 1H, J = 6.4 Hz), 7.44 (t, 1H, J = 7.2 Hz),
7.36 (s, 1H), 6.11 (d, 1H, J = 1.2 Hz), 4.06 (t, 4H, J = 4.4 Hz), 3.89 (t,
4H, J = 5.2 Hz), 3.79 (s, 2H), 3.63 (br t, J = 4.4 Hz), 3.53 (br t, J = 4.4
Hz), 2.76 (t, 2H, J = 6.4 Hz), 2.59 (t, 2H, J = 6.4 Hz), 2.48 (m, 4H),
2.11 (d, 3H, J = 1.2 (Hz), 1.84 (d, 3H, J = 1.2 Hz) ppm. 13C NMR
(100 Hz, CDCl3): δ 199.5, 170.34, 162.5, 160.4, 158.1, 155.6, 149.1,
140.9, 136.5, 132.0, 126.4, 123.8, 123.7, 121.9, 121.6, 113.1, 111.5,
66.8, 57.4, 53.4, 52.9, 52.7, 46.4, 45.1, 41.6, 38.6, 30.9, 27.7, 26.9, 20.8
ppm. MS: m/z 574.2 (M + H). HRMS calcd for C30H37N7O3S
574.2600, found 574.2634.
tert-Butyl 4-((2-Chloro-4-morpholinothieno[3,2-d]pyrimidin-
6-yl)methyl)piperazine-1-carboxylate (1). 2-Chloro-4-
morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde (0.40 g, 1.5
mmol), tert-butylpiperazine 1-carboxylate, and 0.2 mL of acetic acid
were dissolved in 12 mL of dichloroethane. The mixture was stirred at
room temperature for 2 h. NaBH(OAc)3 (0.54g, 2.5 mmol) was added
to the reaction mixture, and the resulting mixture was stirred at room
temperature for 10 h. Then 20 mL of NaHCO3 aqueous solution and
10 mL of DCM were added. The organic layer was separated and dried
over Na2SO4. After removal of solvent, the crude product was
subjected to chromatography on silica gel (eluent, EtOAc/hexane 3:7).
A total of 0.30 g of the title compound was obtained. MS: m/z 454.2
1
(M + H). H NMR (CDCl3, 400 MHz): δ 7.16 (s, 1H), 4.37 (q, J =
4.4 Hz, 4H), 3.90 − 3.78 (m, 6H), 3.46 (t, J = 4.8 Hz, 4H), 2.49 (t, J =
5-(Diethoxyphosphoryl)-4-oxopentanoic Acid (4). To a
solution of diethyl methylphosphonate (0.76g, 5.0 mmol) in 20 mL
of THF at −78 °C was added n-BuLi (2.5 N in hexane, 5.0 mmol)
slowly. The reaction mixture was stirred at −78 °C for 1 h. Succinic
anhydride (0.50 g 5.0 mmol) in 5.0 mL of anhydrous THF was
introduced slowly into the mixture at −78 °C. The reaction mixture
was stirred for 1 h at −78 °C. Then 1 N HCl (5.0 mL) aqueous
solution was added and the mixture was warmed to room temperature.
The THF was then removed under vacuum, and the remaining
aqueous residue was extracted with dichloromethane (3 × 10 mL).
The organic layer was dried over Na2SO4, filtered, and the solvent was
removed. The residue was purified by chromatography on silica gel
(eluent, EtOAc/MeOH 20:1) to provide the acid 4. MS: m/z 253.1
(M + H); 1H NMR (400 MHz, CDCl3): δ 4.15 (4H, m), 3.18 (1H, s),
3.13 (1H, s), 2.95 (2H, t, J = 6.4 Hz), 2.63 (2H, t, J = 6.4 Hz), 1.33
(6H, m) ppm.
tert-Butyl 4-((2-(3-Hydroxyphenyl)-4-morpholinothieno[3,2-
d]pyrimidin-6-yl)methyl)piperazine-1-carboxylate (5). To a
mixture of chloropyrimidine 1 (305 mg, 0.67 mmol), 3-hydrox-
yphenylboronic acid (139 mg, 1.0 mmol), and sodium carbonate (214
mg, 2 mmoL) in degassed toluene/ethanol/water (6 mL/3.6 mL/1.8
mL) was added tetrakis(triphenylphosphine)palladium (51 mg, 0.067
mmol). The reaction mixture was heated to 120 °C for 1 h under
nitrogen, and the solvent was then removed under reduced pressure.
The residue was subject to a quick aqueous EtOAc/water workup and
purified by chromatography on silica gel (EtOAc/heptane v/v 1/1),
giving 238 mg of yellow foamy solid as the title compound (69%). MS:
m/z 512.3 (M + H). 1H NMR (400 MHz, DMSO-d6): δ 9.49 (s, 1H),
7.87−7.81 (m, 2H), 7.38 (s, 1H), 7.26 (t, J = 8.11 Hz, 1H), 6.87−6.83
4.8 Hz, 4H), 1.45 (s, 9H) ppm.
tert-Butyl 4-((2-(1H-Indazol-4-yl)-4-morpholinothieno[3,2-
d]pyrimidin-6-yl)methyl)piperazine-1-carboxylate (2). To a 10
mL microwave tube were added Boc-piperazine 1 (91 mg, 0.20 mmol),
(1H-indazol-4-yl)boronic acid (40 mg, 0.25 mmol), Na2CO3 (84 mg,
0.79 mmol) with 1.6 mL of degassed N,N-dimethylacetamide, and 0.4
mL of degassed water, followed by Pd(PPh3)2Cl2 (8.2 mg, 0.01
mmol). The whole system was repurged with nitrogen and then
heated in a microwave reactor at 120 °C for 1 h. The reaction mixture
was diluted with water and extracted with dichloromethane (DCM).
The combined organic layers were dried over anhydrous Na2SO4.
After the removal of solvent under reduced pressure, the crude
material was purified by silica gel column chromatography (DCM/
MeOH v/v 30/1) to afford compound 2 (78 mg, 73%) as slight yellow
solid. 1H NMR (CDCl3, 400 MHz): δ 9.0 (s, 1H), 8.26 (d, J = 6.8 Hz,
1H), 7.6−7.5 (m, 3H), 7.38 (s, 1H), 4.15−4.06 (m, 4H), 3.98−3.75
(m, 6H), 3.48 (t, J = 4.6 Hz, 4H), 2.60−2.43 (m, 4H), 1.46 (s, 9H)
ppm. MS: m/z 536.1 [M + H].
1-(4-((2-(1H-Indazol-4-yl)-4-morpholinothieno[3,2-d]-
pyrimidin-6-yl)methyl)piperazin-1-yl)-6-methylhept-5-ene-
1,4-dione (3). To a stirred solution of compound 2 (54 mg, 0.1
mmol) in 1 mL of DCM and 1 mL of MeOH was added 4 M HCl in
dioxane (2 mL), and the resulting reaction mixture was stirred at room
temperature for 1 h. The solvent was then removed under reduced
pressure, and the residue was dried in vacuum and used directly for the
following step. MS: m/z 436.1 (M + H).
6-Methyl-4-oxohept-5-enoic acid (17 mg, 0.11 mmol) and carbon-
yldiimidazole (17 mg, 0.11 mmol) were mixed in 0.5 mL of N,N-
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(m, 1H), 4.00−3.93 (m, 4H), 3.87 (s, 2H), 3.84−3.77 (m, 4H), 3.39−
3.33 (m, 4H), 2.44 (s, 4H), 1.39 (s, 9H) ppm. 13C NMR (100 Hz,
CDCl3): δ 159.8, 158.9, 157.6, 156.5, 154.7, 149.7, 139.2, 129.5, 123.0,
120.1, 117.6, 115.0, 113.0, 79.9, 66.7, 57.5, 52.8, 46.3, 28.4 ppm.
HRMS calcd for C26H34N5O4S 512.2331, found 512.2332 (M + H).
Diethyl (5-(4-((2-(3-Hydroxyphenyl)-4-morpholinothieno-
[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2,5-dioxopentyl)-
phosphonate (6). Boc-piperazine 5 (360 mg, 0.7 mmol) was
dissolved in 3 mL of 4 N HCl dioxane solution and dichloromethane
(5 mL), and the reaction was continued for 3 h at room temperature.
After removal of solvents, a white solid (350 mg) was obtained and
used directly for the next step. MS: m/z 412.1 (M + H).
26.9, 23.2 ppm. MS: m/z 1169.4 (M + H); 1167.6 (M-H). HRMS
Calcd for C59H81N10O11S2 1169.5528, found 1169.5529.
Assays. Mass Spectrometry. Mass spectrometry was used to
confirm that 3 covalently bonds to PI3Kα but not to the other class I
isoforms of PI3K. 3 was incubated with PI3Kα (Millipore, 14-602),
PI3Kβ (Millipore, 14-603), PI3Kδ (Millipore, 14-604), PI3Kγ (BPS
bioscience, BPS-40625) at a 10:1 compound-to-enzyme molar ratio at
room temperature for 1 h. After the incubation, 10 mL of 0.2% TFA in
water was added to each sample. Protein enrichment was achieved
with a C4-packed ZipTip (Millipore), direct spotting onto a MALDI
target plate, and combination with equal volume of sinapinic acid (10
mg/mL in 50% acetonitrile) as the desorption matrix. Once dry, the
samples were analyzed on an AB Sciex 4800 MALDI TOF/TOF
instrument.
Trypsin Digest. After the reaction of PI3Kα with 3 as described
above, the protein was separated by electrophoresis on a 4−12% Bis-
Tris gel and stained with a Coomassie blue dye. The PI3Kα band was
then excised from the gel and subjected to a standard in-gel trypsin
digest. Peptide extract was then spotted directly on a MALDI target
plate and combined with an equal volume of α-cyano-4-hydroxycin-
namic acid (5 mg/mL in 80% acetonitrile) as the desorption matrix.
Once dried, the spots were analyzed on an AB Sciex 4800 MALDI
TOF/TOF instrument in both MS and MS/MS modes to confirm
modification of the target amino acid for 3. The targeted Cys862 in
peptide NSHTIMQIQCK was clearly modified.
Phosphonate 4 (750 μL, 0.25 M acetonitrile solution, 187 μmol)
was treated with carbonyldiimidazole (30 mg, 187 μmol) in 1 mL of
N,N-dimethylacetamide for 30 min, followed by addition of the de-Boc
intermediate obtained above (27.5 mg, 50 μmol). After being stirred
for an additional 30 min, the reaction mixture was concentrated and
the residue was purified by preparative HPLC, giving 15 mg of white
1
powder after lyophilization (47%). H NMR (400 MHz, CD3OD): δ
7.71 (t, J = 5.3 Hz, 2H), 7.23 (s, 1H), 7.18 (t, J = 7.9 Hz, 1H), 6.79
(ddd, J = 8.1, 2.5, 1.0 Hz, 1H), 4.09−4.00 (m, 4H), 3.98 (dd, J = 8.9,
4.4 Hz, 4H), 3.82 (s, 2H), 3.80−3.74 (m, 4H), 3.56−3.48 (m, 4H),
2.81 (t, J = 6.3 Hz, 2H), 2.60−2.49 (m, 4H), 2.49−2.42 (m, 2H), 1.23
(t, J = 7.1 Hz, 6H) ppm. 13C NMR (100 Hz, CD3OD): δ 202.8 (d, J =
6.2 Hz), 172.2, 163.0, 161.4, 159.0, 158.7, 151.9, 140.8, 130.4, 123.6,
120.5, 118.3, 116.1, 114.2, 67.8, 64.1 (d, J = 6.4 Hz), 58.0, 53.9 (d, J =
25.6 Hz), 47.7, 46.6, 43.0, 39.8 (d, J = 2.4 Hz), 28.0, 16.6 (d, J = 6.2
Hz) ppm. MS: m/z 646.5 (M + H). HRMS calcd for C30H41N5O7S
646.2464, found 646.2466.
(E)-tert-Butyl 4-(6-(4-((2-(3-Hydroxyphenyl)-4-
morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-
yl)-3,6-dioxohex-1-enyl)benzylcarbamate (7). A mixture of the
phosphonate 5 (13 mg, 20 μmol), tert-butyl 4-formylbenzylcarbamate
(10 mg, 40 μmol), potassium carbonate (40 mg) in 1 mL of N,N-
dimethylacetamide, and 100 μL of water was heated at 70 °C for 4 h.
After filtration, the reaction mixture was purified by preparative HPLC,
giving 10 mg of desired enone 7 as a white solid. 1H NMR (400 MHz,
DMSO-d6): δ 9.49 (s, 1H), 7.87−7.82 (m, 2H), 7.67 (d, J = 8.0 Hz,
2H), 7.58 (d, J = 16.3 Hz, 1H), 7.39 (s, 2H), 7.28 (dd, J = 8.3, 2.7 Hz,
3H), 6.89 (d, J = 16.2 Hz, 2H), 4.15 (d, J = 6.1 Hz, 2H), 3.96 (dd, J =
19.8, 14.9 Hz, 6H), 3.86−3.78 (m, 4H), 3.50 (d, J = 22.2 Hz, 4H),
3.32 (d, J = 9.5 Hz, 2H), 2.90 (d, J = 6.4 Hz, 2H), 2.62 (s, 2H), 2.44
(s, 2H), 1.37 (d, J = 24.0 Hz, 9H) ppm. 13C NMR (100 Hz, MeOD):
δ 199.3, 170.3, 162.2, 160.0, 157.8, 156.6, 156.0, 155.9, 149.2, 142.6,
139.7, 133.5, 129.5, 128.6, 127.9, 126.0, 123.4, 120.0, 117.4, 115.1,
113.1, 79.8, 66.8, 57.4, 53.0, 46.3, 45.3, 41.7, 35.4, 28.4, 27.1 ppm. MS:
m/z 727.3 (M + H). HRMS calcd for C39H47N6O6S 727.3278, found
727.3276.
GSH Reactivity. GSH reactivity was assessed by incubating 10 μM
3 at 37 °C in PBS at pH 7.4 with or without GSH (5 mM). At various
designated time points, 50 μL of the reaction mixture was diluted with
50 μL of internal standard (1 μg/mL carbutamide in acetonitrile),
injected onto a C8 column (Agilent Technologies, Zorbax, 3.5 μm, SB-
C18, 2.1 mm × 30 mm), and analyzed with an AB Sciex QTrap 4000
mass spectrometer. Parent recovery was assessed and calculated as
percent of the time zero value.
Plasma Protein Bonding. Human plasma, obtained from
Bioreclamation (Hicksville, NY), was subjected to albumin depletion
using the Pierce Blue albumin removal kit (catalog no. 89845, Thermo
Scientific, Rockford, IL) according to product procedures. The
albumin-depleted plasma was then diluted to a final concentration of
1 mg/mL. It is important to note that albumin was depleted but not
completely removed. A 10 mM DMSO stock of 3 was diluted 1:10
into 10 mg of the albumin-depleted human plasma. The reaction was
carried out in a 96-well plate format in a 37 °C incubator. At the end
of the incubation, 10 μL aliquots of the samples were diluted with 10
μL of 0.2% TFA and then pipetted with C4 ZipTips (Millipore)
directly onto a MALDI target plate, using sinapinic acid as the
desorption matrix (10 mg/mL in 0.1%TFA in acetonitrile/water 50:50
v/v) and ovalbumin as the internal standard. Samples were analyzed
on an ABSciex 4800 MALDI TOF/TOF mass spectrometer.
pAktS473 Immunoblot. SKOV3 cells were plated in SKOV3
growth medium (DMEM supplemented with 10% FBS and penicillin/
streptomycin) at a density of 4 × 105 cells per well of 12-well plates.
Twenty-four hours later the medium was removed and replaced with 1
mL of medium containing test compound in 0.1% DMSO and cells
were returned to the incubator for 1 h. At the end of the hour, the
medium was removed and the cells were washed with PBS, then lysed
and scraped into 30 μL of cell extraction buffer (Biosource, Camarillo,
CA) plus Complete protease inhibitor and PhosStop phosphatase
inhibitor (Roche, Indianapolis, IN). Cell debris was spun down at
13000g for 1 min, and the supernatant was taken as the cell lysate.
Protein concentration of the lysate was determined by BCA Assay
(Pierce Biotechnology, Rockford, IL), and 50 μg of protein was loaded
per well onto a NuPAGE Novex 4−12% Bis-Tris gel (Invitrogen,
Carlsbad, CA) and then transferred to Immobilon PVDF-FL
(Millipore, Billerica, MA).
Biotinylated Covalent Probe (8). To 7.5 mg of enone 7 in 1 mL
of dichloromethane was added 1 mL of trifluoroacetic acid. After the
mixture was stirred for 30 min, the solvent was removed under
reduced pressure, giving de-Boc intermediate with m/z 627.3 (M +
H). Then 1 mL of N,N-dimethylacetamide, 60 μL of DIPEA, and 9.0
mg of commercially available N-biotinyl-NH(PEG)2-COOH-DIPEA
(20 atoms) (Novabiochem catalog no. 851029) were added, followed
by 9 mg of HATU. After being stirred for an additional 30 min, the
crude mixture was purified by preparative HPLC, giving 9 mg of
1
desired compounds as a white powder after lyophilization. H NMR
(400 MHz, CD3OD): δ 7.76 (s, 1H), 7.67 (br t, 2H, J = 8.4 Hz), 7.60
(m, 2H), 7.44 (t, 1H, J = 8.0 Hz), 7.32 (d, 2H, J = 8.4 Hz), 7.13 (dd,
1H, J = 2.8, 16.4 Hz), 6.83 (d, 1H, J = 16.4 Hz), 4.66 (s, 2H), 4.48 (m,
2H), 4.38 (s, 2H), 4.30 (m, 4H), 3.92 (m, 4H), 3.83 (m, 2H), 3.62 (m,
4H), 3.57 (m, 4H), 3.50 (m, 4H), 3.15−3.30 (m, 8H), 3.70 (br t, 2H, J
= 6.4 Hz), 2.90 (m, 2H), 2.67−2.76 (m, 4H), 2.15−2.30 (m, 8 H),
1.90 (m, 4H), 1.70−1.80 (m, 8H), 1.60 (m. 6H,), 1.40 (m, 4H) ppm.
13C NMR (100 Hz, CD3OD): δ 201.3, 175.9, 175.3, 173.0, 166.0,
160.7, 160.3, 159.6, 158.1, 157.4, 152.2, 150.3, 144.0, 143.1, 134.8,
133.6, 131.6, 130.0, 129.7, 129.1, 126.8, 122.6, 122.2, 121.5, 120.3,
118.3, 116.4, 115.6, 71.5, 71.2, 69.9, 67.4, 63.4, 61.6, 57.0, 55.1, 53.2,
52.9, 43.8, 41.0, 40.8, 37.8, 36.8, 36.3, 36.2, 36.1, 30.4, 29.8, 29.5, 27.5,
The blot was blocked in Odyssey blocking buffer (Li-Cor
Biosciences, Lincoln, NE) for 1 h and then incubated overnight at 4
°C with mouse anti-Akt (no. 2920) and rabbit anti-phospho-Akt
(Ser473) (no. 9271); Cell Signaling Technology, Beverley, MA)
antibodies, both diluted 1:1000 in PBS/Odyssey Buffer (1:1) + 0.1%
Tween-20. The blots were washed 3 times for 5 min each in PBS +
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0.2% Tween-20 and then incubated for 1 h at room temperature with
fluorescently labeled secondary antibodies (Li-Cor) diluted 1:10000 in
PBS/Odyssey buffer (1:1) + 0.1% Tween-20.
The blots were washed 2 times for 5 min each in PBS + 0.2%
Tween-20, once in distilled water, then scanned on an Odyssey
machine (Li-Cor). Band intensity was determined using the Odyssey
software, and the phopho-Akt signal was normalized to total Akt
within samples, then expressed as a percentage of the untreated
phospho-Akt signal.
Demonstration of Prolonged Duration of Inhibition. SKOV3
cells were plated in SKOV3 growth medium (DMEM supplemented
with 10% FBS and pen/strep) at a density of 2.5 × 104 cells per well of
Costar no. 3603 black 96-well clear flat-bottom plates. Plates were set
up in quadruplicate with one plate each for the 0, 1, 6, and 24 h time
points.
curve from 1000 to 4 nM. The cells were incubated for 96 h and then
developed with Cell Titer Glo.
The cell numbers at the end of the assay were determined using the
standard curve generated at the start of the assay. Growth inhibition
was calculated using the following formulas, and GI50 values were
determined by plotting the percent growth inhibition vs log of the
compound concentration in GraphPad:
T − T
0
% growth = 100 ×
C − T
0
where T is the cell number at end of assay, T0 is the cell number at
start of assay (5000), C is the mumber of cells in DMSO controls at
end of assay, and
% growth inhibition = 100 − % growth
Twenty-four hours later the medium was removed and replaced
with 100 μL of medium containing test compound or DMSO as a
control and cells were returned to the incubator for 1 h. At the end of
the hour, the medium was removed and the cells were washed 2 times
with PBS. The PBS was removed from three of the plates, replaced
with 100 μL of growth medium, and the plates were returned to the
incubator. The fourth plate was taken as the “0”-hour time point and
developed as described for in-cell Western or Western blot. Thirty
minutes after the first wash, the medium was removed from the
remaining plates, replaced with 100 μL of fresh growth medium, and
then the plates were returned to the incubator. At 1 h after the first
wash, one plate was taken as the 1 h time point and developed as an
in-cell Western. The remaining two plates were washed two more
times at 1 h intervals and developed as in-cell Western or Western blot
at 6 and 24 h after the first wash.
In-Cell Western. Treated SKOV3 cells were washed once with
PBS, then fixed for 20 min at room temperature in 4% formaldehyde
in PBS. The formaldehyde was removed, and cells were washed 5
times for 5 min each with 100 μL of permeabilization buffer (PBS +
0.1% Triton X-100) at room temperature with gentle shaking. The last
wash was removed and replaced with 150 μL of Odyssey blocking
buffer (Li-Cor, Lincoln, NE) and incubated for 90 min at room
temperature with gentle shaking.
The blocking buffer was then replaced with 50 μL of primary
antibody mix (rabbit anti-phospho-Akt(Ser473) at 1:100 (Cell
Signaling Technology, Boston, MA) and mouse anti-tubulin at
1:5000 (Sigma Aldrich, St. Louis, MO) diluted in Odyssey blocking
buffer) and incubated overnight at room temperature with gentle
shaking.
The next morning, the antibody mix was removed and the wells
were washed 5 times for 5 min each with PBS + 0.1% Tween-20. The
last wash was replaced with 50 μL of secondary antibody mix (goat
anti-rabbit-IRDye-680 and goat anti-mouse-IRDye-800 (Li-Cor), both
diluted 1:1000 in Odyssey blocking buffer + 0.2% Tween-20) and
incubated for 1 h at room temperature with gentle shaking. The
antibody mix was removed, and the wells were washed 5 times for 5
min each in PBS + 0.1% Tween-20, then 1 time with doubly distilled
H2O.
Pharmacokinetic Study of 3. Fasted male C57BL/6 mice were
dosed intraperitoneally (3 animals/time point) at 10 mg/kg using a
dosing formulation consisting of 15% Solutol HS 15/5% DMSO/80%
PBS. Blood samples (300 μL) were collected via terminal cardiac
puncture and placed into chilled tubes containing EDTA as the
anticoagulant. The samples were centrifuged at 4 °C at 13 000 rpm for
5 min, and plasma was collected and stored frozen until analysis. 3 was
extracted from the plasma samples by protein precipitation, and the
plasma concentration of 3 was assessed by LC−MS/MS using an API
4000 QTrap mass spectrometer. The bioanalytical method details are
shown in Table S2, and the pharmacokinetic data are shown in Table
S3 in Supporting Information.
The calibration curve of 3 was constructed using standards ranging
from 1 to 5000 ng/mL. The regression analysis of 3 was performed by
plotting the peak area ratio of 3 over internal standard (y) against the 3
concentration (x) in ng/mL. Pharmacokinetic analysis was conducted
using Analyst software (version 1.4.2; Life Technologies; Carlsbad,
CA) for LC−MS/MS data management and WinNonlin (version 5.2;
Pharsight Corporation; Mountain View, CA) for PK analysis. Mean
plasma concentration values for each time point were used to generate
time to achieve peak plasma concentration [Tmax (h)], plasma terminal
half-life [t1/2 (h)], peak plasma concentration [Cmax (ng/mL)], and the
area under the plasma concentration−time curve [AUC ((h·ng/mL)].
In Vivo Pharmacodynamic Evaluation of the PI3Kα Covalent
Inhibitor. Female nu/nu (n = 3/group) were administered compound
(GDC-0941 or 3) delivered ip at 100 mg/kg once daily for 5
consecutive days. After delivery of the last dose, spleens from treated
animals were harvested at 1, 4, and 24 h time points. Spleens were
immediately frozen in liquid nitrogen. Samples were stored at −80 °C
until processing for homogenates. Homogenates were made by adding
approximately 100 μL of spleen sample to a Precellys homogenizing
tube (Precellys, Montigny, France) containing 300 μL of cell
extraction buffer (Biosource, Camarillo, CA) plus Complete protease
inhibitor and PhosStop phosphatase inhibitor (Roche, Indianapolis,
IN) and kept on ice. The sample was homogenized in a Precellys 24
homogenizer for 15 s followed by centrifugation at 16000g for 20 min
at 4 °C. The supernatant was moved to a new tube, and the protein
concentration was determined by BCA Assay (Pierce Biotechnology,
Rockford, IL).
The plates were scanned on an Odyssey machine (Li-Cor) with a 3
mm focus offset at an intensity of 8 in both channels, and the data
were analyzed using the Odyssey software.
An amount of 150 μg of protein sample was added to a 0.2 mL tube
and the volume brought up to 100 μL with IP buffer from the protein
A/G plate IP kit (Pierce Biotechnology, Rockford, IL). The covalent
probe compound, 8, was added at 1 μM, and the tube was incubated at
room temperature with rocking for 1 h. Protein A/G coated wells from
the protein A/G plate IP kit were washed 3× with 200 μL of IP buffer
before coating with 4 μL of rabbit anti-p110α antibody (no. 4249, Cell
Signaling Technology, Danvers, MA) plus 36 μL of IP buffer per well.
After incubating at room temperature with shaking for 1 h, the wells
were washed 5× with 200 μL of IP buffer and the protein samples,
preincubated with 8, were added to the wells. The wells were
incubated overnight at 4 °C with shaking. The wells were then washed
5× with 200 μL of IP buffer. The immuoprecipitate was eluted from
the plate with 40 μL of Pierce elution buffer (Pierce Biotechnology,
Rockford, IL), and the eluate was moved to a 1.5 mL tube containing 4
Cell Proliferation Assays and GI50 Determinations in SKOV3
and MCF-7 Cells. SKOV3 cells or MCF-7 cells were plated in
SKOV3 proliferation assay medium (DMEM supplemented with 5−
10% FBS and pen/strep) at a density of 5000 cells in 180 μL volume
per well in Costar no. 3610 white 96-well clear flat-bottom plates and
incubated overnight in a humidified 37 °C incubator. A standard curve
ranging from 10 000 to 50 000 cells was set up in a separate plate and
allowed to adhere to the plate for 4−6 h, at which time the plate was
developed using Cell Titer-Glo (Promega, Madison, WI) according to
the manufacturer’s instructions.
The next morning, 3-fold compound dilutions ranging from 10 000
to 40 nM were prepared in proliferation medium containing 1%
DMSO. Then 20 μL of each dilution was added to the SKOV3 or
MCF-7 cells plated the previous day, resulting in a dose−response
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μL of Pierce neutralization buffer (Pierce Biotechnology, Rockford,
IL). The proteins were separated by electrophoresis and immuno-
blotted as described above. p110α was detected using an anti-p110α
antibody (Epitomics, Burlingame, CA) diluted 1:2500 in PBS/
Odyssey buffer (1:1) + 0.1% Tween-20. The blot was subsequently
incubated with a fluorescently labeled secondary antibody, goat anti-
rabbit-IRDye800 (Li-Cor) diluted 1:10000 in PBS/Odyssey buffer
(1:1) + 0.1% Tween-20, and streptavidin-AlexaFluor-680 (Invitrogen)
diluted 1:1000 to detect the biotinylated probe, The streptavidin
signal, detecting covalent probe binding, was normalized to total
p110α signal within samples, then expressed as a percentage of the
vehicle treated samples.
Kinase Selectivity Panel. 3 was tested in a panel of 10 lipid
kinases at the Reaction Biology Corporation (Malvern, PA). Briefly,
compound was tested in a 10-concentration IC50 curve with 3-fold
serial dilution starting at 1 μM. Reactions were carried out at 10 μM
ATP. An HTRF assay format was used for PI3Kα, PI3Kβ, PI3Kγ, and
PI3Kδ; ADP-GLO assay format was used for other kinases. The
following substrates were used: for HTRF, phosphatidylinositol 4,5-
bisphosphate; for SPHK1 and SPHK2, sphingosine; for other ADP-
GLO enzymes, phosphatidylinositol.
For general kinase selectivity, 3 was run in a kinase selectivity panel
at Reaction Biology Corporation (Malvern, PA) using HotSpot
technology and radioisotope-based P81 filtration. 3 was dissolved in
pure DMSO to the final 1 μM test concentration. Substrates for the
various kinases tested against 3 were prepared fresh daily in reaction
buffer. Any required cofactors were then added to the substrate
solution followed by kinase addition and preincubated for 30 min at
room temperature. 33P-ATP (10 μM) was delivered into the reaction
mixture to initiate the reaction, and reaction continued for 2 h at room
temperature. The reaction was terminated, and any unreacted
phosphate was washed away using 0.1% phosphoric acid prior to
detection utilizing a proprietary technology (Reaction Biology Corp.;
Malvern, PA, U.S.). The study was performed in duplicate, and 10 μM
staurosporine, a nonselective, ATP-competitive kinase inhibitor, was
used as the positive control.
Molecular Modeling of 3. The binding mode of 3 to PI3Kα was
obtained through molecular modeling to the X-ray structure of PI3Kα
(p110α/p85α complex, PDB code 2RD0). We initially superimposed
the complex structures of PI3Kγ (GDC-0941, PDB code 3DBS) and
PI3Kα, then docked GDC-0941 into the PI3Kα protein. The key
hinge binding interactions of the thienopyrimidine scaffold between
PI3Kα and PI3Kγ are almost identical. We identified Cys862 as being
unique to PI3Kα and determined it to be approximately 10 Å from the
thienopyrimidine of GDC-0941. Through an iterative structure-based
drug design process where we explored variations in linker length and
electrophilic functionality, we identified 3. The covalent bond between
the thiol (Cys862) and 3 was formed in silico through a local
minimization using CHARMM force field in Discovery Studio
(Discovery Studio Modeling Environment, release 2.1; Accelrys Software
Inc.: San Diego, CA, 2008).
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 781-541-3700. Fax: 781-541-5101. E-mail: mnacht@
celgene.com.
Author Contributions
All authors contributed equally.
Notes
The authors declare the following competing financial
interest(s): The authors are employees of Celgene (except D.
Bhavsar, M. P. Sheets, W. Westlin).
All authors except for D. Bhavsar, M. P. Sheets, and W. Westlin
are shareholders in Celgene.
ACKNOWLEDGMENTS
■
We thank Proteros Biostructures for assistance in solving the
complex of PI3Kα with compound 3.
ABBREVIATIONS USED
■
PI3K, phosphoinositol 3-kinase; PTEN, phosphate and tensin
homologue; GSH, glutathione; ip, intraperitoneally
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ASSOCIATED CONTENT
■
S
* Supporting Information
Chemical structure of the covalent probe 8; mass spectrometry
on 3 with PI3K family members; specificity of 3 toward PI3Kα
vs other kinases; lack of reactivity of 3 to nonspecific thiols
including glutathione and plasma proteins; inhibition of PI3Kα
signaling in SKOV3 cells by 3; specificity of the covalent probe
8 for PI3Kα vs PI3Kβ in vitro; X-ray crystallography parameters
of 3 complexed with PI3Kα. This material is available free of
charge via the Internet at http://pubs.acs.org.
Accession Codes
The structure of 3 complexed with PI3Kα has been deposited
in the Protein Data Bank, and the PDB code is 3zim.
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