From the
Several natural and unnatural inositol phosphates and analogues
were analyzed for their ability to inhibit the in vitro phosphatidylinositol 3-kinase (PI 3-kinase) activity
immunoprecipitated from a leukemic T cell line by a p85 monoclonal
antibody. A 3-position ring-modified analogue of
D-myo-inositol 1,4,5-trisphosphate
(Ins(1,4,5)P
Phosphatidylinositol 3-kinase (PI 3-kinase)¹(¹)
is a heterodimer of a 85-kDa regulatory subunit
(1) and a 110-kDa catalytic subunit
(2) that mediates the
formation of D-3-phosphatidylinositol lipids by transferring
the terminal phosphate of ATP to the D-3-position on inositol
head groups of phosphatidylinositol (PtdIns), phosphatidylinositol
4-monophosphate (PtdIns(4)P), and phosphatidylinositol 4,5-bisphosphate
to yield phosphatidylinositol 3-monophosphate (PtdIns(3)P),
phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol
3,4,5-trisphosphate, respectively
(3) . PI 3-kinase is coupled to
receptors containing intrinsic or associated protein-tyrosine kinase
activities as well as to G protein-associated receptors (reviewed in
Refs. 3 and 4), and the D-3-phosphatidylinositols are thought
to act as regulatory molecules utilized by a number of receptors
involved in mitogenic and/or chemotactic responses in
cells
(3, 4) .
Although its precise cellular role
remains unknown, phosphatidylinositol 3,4,5-trisphosphate is believed
to be the physiologically relevant lipid
(3) . Clarification of
the precise function of such lipids is still hampered by a lack of
agents to manipulate the activity of PI 3-kinase. Effective inhibitors
of this enzyme may help to define the role of PI 3-kinase and its
metabolic products in cells, but to date, only a few inhibitors have
been described. Generally, these are derived from natural products,
namely the fungal metabolite
wortmannin
(5, 6, 7, 8) , the
structurally related but distinct compound demethoxyviridin
(9) ,
the bioflavanoid quercetin (10), as well as certain synthetic
chromones
(11) . To date, wortmannin, which binds to the
ATP-binding site of the p110 subunit
(6) , is the most effective
PI 3-kinase inhibitor, being active at nanomolar concentrations.
However, wortmannin at micromolar concentrations has been shown to
inhibit other enzymes such as phospholipase D
(12, 13, 14) and myosin light chain kinase
(15) .
The PI
3-kinase phosphorylation target site on phosphatidylinositols is
similar to that on myo-inositol 1,4,5-trisphosphate
(Ins(1,4,5)P
Several other inositol
phosphate analogues can inhibit 5-phosphatase, albeit with much lower
potency. These include
L-Ins(1,4,5)P
L-chiro-Ins(2,3,5)P
Thus, there appears to be an
absolute requirement for axial hydroxyl groups at both 2- and
3-hydroxyl positions (D-Ins(1,4,5)P
The
observation that the inhibition of PI 3-kinase by
L-chiro-Ins(2,3,5)P
L-chiro-Ins(2,3,5)P
This study suggests that such
simplified structures may also represent lead compounds in the
development of PI 3-kinase inhibitors. Clearly, a future strategy to
exploit these observations could include synthesis of related analogues
possessing part or all of the missing diacylglycerol element. This
should enhance recognition of the analogues by PI 3-kinase and
potentially enhance potency of the inhibitory effect on PI 3-kinase
activity. In the development of second generation analogues based on
these lead compounds, it should also be an important consideration to
avoid dual specificity, which may compromise their use as specific
inhibitors of PI 3-kinase or Ins(1,4,5)P
K
The presented representative data were determined by
double-reciprocal plots. K
We thank A. Riley and Dr. D. Lampe for provision of
inositol polyphosphate analogues and Dr. R. Eisenthal for helpful
discussions.
), L-chiro-inositol
2,3,5-trisphosphate (L-chiro-Ins(2,3,5)P
)
and its phosphorothioate analogue, L-chiro-inositol
2,3,5-trisphosphorothioate, as well as the analogue benzene
1,2,4-trisphosphate induced reversible inhibition of PI 3-kinase
activity, which correlated with decreased V
but
unchanged K
values for PI 3-kinase. Other
inositol phosphates, including D- and
L-Ins(1,4,5)P
, D-myo-inositol
1,3,4,5-tetrakisphosphate, the enantiomers of myo-inositol
1,3,4-trisphosphate, DL-myo-inositol
1,4,6-trisphosphate (DL-Ins(1,4,6)P
), and
DL-scyllo-inositol 1,2,4-trisphosphate
(DL-scyllo-Ins(1,2,4)P
), did not inhibit
PI 3-kinase activity under identical conditions.
L-chiro-Ins(2,3,5)P
closely resembles
Ins(1,4,5)P
and D-Ins(1,4,6)P
except
for a difference in the orientation of a single hydroxyl group at
either the equivalent 3-OH or 2-OH position of Ins(1,4,5)P
,
respectively. Similarly, L-chiro-Ins(2,3,5)P
resembles D-scyllo-Ins(1,2,4)P
, but
has a different orientation of both the equivalent 3-OH and 2-OH
positions. Since Ins(1,4,5)P
,
DL-Ins(1,4,6)P
, and
DL-scyllo-Ins(1,2,4)P
did not inhibit PI
3-kinase activity, this suggests that the orientation of the two
hydroxyl groups at the 2- and 3-positions plays a pivotal role in the
inhibitory action of inositol phosphate analogues on PI 3-kinase
activity. Thus, inositol phosphate analogues inter alia are
shown for the first time to inhibit PI 3-kinase and may be useful tools
for determining the function of PI 3-kinase and its substrate binding
specificities.
) 3-kinase in that Ins(1,4,5)P
3-kinase phosphorylates the phospholipase C metabolic product
Ins(1,4,5)P
(Fig. 1, compound 1), also at the
D-3-position of the inositol ring
(16) .
Ins(1,4,5)P
3-kinase is distinct from PI 3-kinase and has
been purified from rat brain
(17, 18) , bovine
brain
(19) , pig aortic muscle
(20) , and
platelets
(21) . Ins(1,4,5)P
3-kinase activity has
been associated with polypeptides of 93
(20) , 70
(21) ,
53
(17) , 52
(19) , 38
(19) and 35
(19) kDa,
and it has thus been suggested that multiple isoforms of this enzyme
occur. However, multiple forms of Ins(1,4,5)P
3-kinase may
be due to calpain-dependent proteolysis of the enzyme
(22) rather than to the existence of isoforms. We have adopted a
novel approach in the search for PI 3-kinase inhibitors by analyzing
the effect on PI 3-kinase activity of natural and unnatural inositol
phosphates, which may compete with the D-3-phosphorylation
site on the inositol head group of phosphatidylinositols for substrate
recognition by PI 3-kinase. Hence, we reasoned that small molecule
inositol phosphate analogues, which have been reported to inhibit the
transfer of phosphate onto Ins(1,4,5)P
catalyzed by
Ins(1,4,5)P
3-kinase
(23) , may additionally act as
leads for compounds that inhibit the phosphorylation of
phosphatidylinositols by PI 3-kinase.
Figure 1:
Structures of Ins(1,4,5)P
(compound 1), L-chiro-Ins(2,3,5)P
(compound 2), L-chiro-Ins(2,3,5)PS
(compound 3), D-Ins(1,4,6)P
(compound 4),
D-scyllo-Ins(1,2,4)P
(compound 5), and
Bz(1,2,4)P
(6).
We report here the first
evidence that chemically synthesized analogues of Ins(1,4,5)P that act as inhibitors of Ins(1,4,5)P
3-kinase and
possess an inverted 3-hydroxyl group, namely
L-chiro-inositol 2,3,5-trisphosphate
(L-chiro-Ins(2,3,5)P
) (Fig. 1,
compound 2)
(23, 24) and
L-chiro-inositol 2,3,5-trisphosphorothioate
(L-chiro-Ins(2,3,5)PS
) (Fig. 1,
compound 3)
(23, 25) , can also inhibit PI
3-kinase. In addition, we report an improved synthesis of benzene
1,2,4-trisphosphate (Bz(1,2,4)P
) (Fig. 1, compound 6)
(26) , a loosely structurally related analogue of
Ins(1,4,5)P
, and demonstrate that it also inhibits PI
3-kinase.
Materials
Chemically synthesized
Ins(1,4,5)P and D-myo-inositol
1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P
) were from the
University of Rhode Island Chemistry Group. p85 monoclonal antibodies
(mAbs) were gifts from Dr. Cantrell (Imperial Cancer Research Fund,
London). ATP, wortmannin, and phosphatidylinositols (soybean PtdIns and
bovine PtdIns(4)P) were purchased from Sigma (Poole, Dorset, United
Kingdom). Wortmannin was dissolved in ethyl acetate to a concentration
of 20 mM and stored in aliquots at
20 °C in the
dark. All other reagents were of analytical grade and were purchased
from Sigma and Aldrich. TLC was performed on Silica Gel 60 F (Merck)
with detection by UV light or phosphomolybdic acid. Flash column
chromatography was performed on Silica Gel SORBSIL C60. Ion-exchange
chromatography was performed using a Pharmacia Biotech medium pressure
ion-exchange chromatograph, Q-Sepharose, and a gradient of
triethylammonium bicarbonate. ¹H NMR spectra (internal
Me
Si reference) were recorded on a Jeol JMN-GX 270 or EX
400 spectrometer.
P NMR spectra (external
H
PO
reference) were recorded on a Jeol EX 400
NMR spectrometer. Chemical shift(s) are reported as negative when
downfield from H
PO
.
C NMR spectra
(internal CDCl
reference) were recorded on a Jeol JMN-GX
270 spectrometer. Mass spectra were recorded at the Engineering and
Physical Sciences Research Council Mass Spectrometry Service (Swansea,
UK). Microanalysis was carried out by the Microanalysis Service at the
University of Bath.
Cell Culture
The leukemic T cell line Jurkat was
grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal
calf serum, 50 µg/ml streptomycin, and 50 units/ml penicillin at 37
°C (27).
Chemical Synthesis of Inositol
Phosphates
Ins(1,4,5)P analogues, including
DL-myo-inositol 1,4,6-trisphosphate
(DL-Ins(1,4,6)P
)
(28) ,
L-chiro-Ins(2,3,5)P
(23, 24) ,
L-chiro-Ins(2,3,5)PS
(23, 25), and
L-Ins(1,4,5)P
(29), were synthesized as described,
and the structures of the most important of these are given in
Fig. 1
. References for further inactive analogues are given in
the text. All compounds were purified by ion-exchange chromatography on
Q-Sepharose Fast Flow (Pharmacia Biotech Inc.) using a gradient of
triethylammonium bicarbonate, pH 8.0, quantified using the Briggs
phosphate assay
(30) and used as their triethylammonium salts.
All compounds showed satisfactory ¹H and
P NMR
and mass spectrometric data.
Synthesis of
1,2,4-Tris(diethylphospho)benzene
Benzene-1,2,4-triol (252 mg, 2
mmol) was suspended in dry dichloromethane (5 ml) and stirred under a
blanket of nitrogen. Dry N,N-diisopropylethylamine
(2.1 ml, 12 mmol) was added to the suspension, and the solution turned
red in color. The solution was then cooled to 78 °C, and
diethyl chlorophosphite (1.57 ml, 9.0 mmol) was added dropwise, giving
a pale yellow color, which indicated that the hydroxyl groups had been
phosphitylated. The cooling bath was removed, and water (2 ml) was
added to the solution, which was stirred for 30 min. t-Butyl
hydroperoxide (1 ml, 7 mmol; 70% solution in water) was added dropwise,
and the mixture was stirred for 15 min at room temperature. Analysis by
TLC showed a new spot at R
= 0.34.
The solution was diluted with dichloromethane (100 ml) and washed with
water (100 ml), 10% sodium metabisulfite (100 ml), 0.1 M
hydrochloric acid (50 ml), saturated sodium hydrogen carbonate solution
(100 ml), and water (100 ml). The organic layer was dried over
magnesium sulfate and evaporated to give the crude product as an oil.
Flash chromatography (9:1 ethyl acetate/ethanol) gave the pure title
compound as a syrup (yield of 0.79 g, 76%). Found: C, 40.7; H, 6.42;
C
H
O
P
requires C,
40.45; H, 6.18.
(CDCl
, 270 MHz),
1.34-1.40 (18H, m,
BzOP(O)OCH
CH
), 4.17-4.32 (12H,
m, BzOP(O)OCH
CH
), 7.03-7.39 (3H,
m, H-3, H-5, H-6, Bz).
(CDCl
, 68 MHz),
15.53-15.63 (2q, BzOP(O)OCH
CH
),
64.43, 64.53, 64.64 (3t,
BzOP(O)OCH
CH
), 113.29-146.91
ring carbons.
(CDCl
, 162 MHz), 6.86 (dtt,
J = 7.83 Hz),
7.12 (dtt, J =
7.83 Hz),
7.28 (dtt, J = 7.83 Hz).
Synthesis of Benzene
1,2,4-Trisphosphate
1,2,4-Tris(diethylphospho)benzene (274 mg,
528 µmol) was dissolved in dry dichloromethane.
Bromotrimethylsilane (0.836 ml, 6.3 mmol) was added dropwise to the
solution, which was then stirred for 16 h. The solvents were
evaporated, and the residue was stirred with water (1 ml). Final
purification of the compound was by elution from a column of
Q-Sepharose Fast Flow using triethylammonium bicarbonate buffer with a
linear gradient of 0-1 M. The title compound
(Fig. 1, compound 6) eluted between 0.2 and 0.5 M
buffer and after evaporation was obtained as its glassy
triethylammonium salt (yield of 456 µmol, 86%). (D
O, 400 MHz), 6.85 (1H, dd, J = 1.5
Hz, J = 8.85 Hz, H-5, Bz), 7.06 (1H, d, J = 1.5 Hz, H-3, Bz), 7.18 (1H, d, J = 8.85
Hz, H-6, Bz).
(CDCl
, 162 MHz),
3.61 (s),
3.92 (s),
4.28 (s). Accurate mass
spectrum requires the following: (M
H)
= 364.9228. Found: 364.9238.
Cell Lysis and Immunoprecipitation
Jurkat cells
were washed, resuspended in RPMI 1640 medium containing 20 mM
HEPES, aliquoted at 2 10
cells/ml, and incubated at
37 °C for 5 min. Cells were pelleted, and the pellets were lysed in
1 ml of lysis buffer (1% Nonidet P-40, 100 mM NaCl, 20
mM Tris, pH 7.4, 10 mM iodoacetamide, 10 mM
NaF, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml
leupeptin, 1 µg/ml antipain, 1 µg/ml chymostatin, 1 µg/ml
pepstatin A, 1 mM sodium orthovanadate). After centrifugation
at 14,000 rpm, post-nuclear lysates were precleared with protein
G-Sepharose to remove any nonspecific proteins that may have bound to
protein G-Sepharose. This was followed by immunoprecipitation for 2 h
at 4 °C as described
(31) using a p85 mAb coupled to protein
G-Sepharose beads (1 µg/ml of cell lysate).
In Vitro Lipid Kinase and Protein Kinase
Assays
Immunoprecipitates were washed and subjected to in
vitro lipid kinase assays as described
(31) using a lipid
mixture of 100 µl of 0.1 mg/ml PtdIns and 0.1 mg/ml
phosphatidylserine dispersed by sonication in 20 mM HEPES, pH
7.0, 1 mM EDTA. The reaction was initiated by the addition of
a mixture of 20 µCi of [-
P]ATP (3000
Ci/mmol; DuPont NEN, Stevenage, UK) and 100 µM ATP to the
immunoprecipitates suspended in 80 µl of lipid kinase buffer. The
reaction was terminated after 15 min by the addition of 80 µl of 1
N HCl and 200 µl of chloroform/methanol (1:1, v/v). After
centrifugation for 5 min at 14,000 rpm, the lower phase was removed,
dried in vacuo, and redissolved in 50 µl of chloroform.
Phospholipids were then separated by thin-layer chromatography in 2
M 1-propanol/acetic acid (65:35, v/v) developing
solvents
(31) . [
P]PtdInsP was visualized
by autoradiography, recovered from the TLC plate, and quantitated by
scintillation counting (Canberra Packard). HPLC analysis of
glycerophosphoryl derivatives of [
P]PtdInsP was
performed as described
(32) . Alternatively, immunoprecipitates
prepared as described above were washed three times in lysis buffer and
twice in protein kinase assay buffer (100 mM NaCl, 25
mM HEPES, pH 7.4, 10 mM MgCl
, 5
mM MnCl
, 100 µM sodium orthovanadate)
as described
(31) . Assays were initiated with 20 µl of
protein kinase buffer containing 10 µM ATP and 10 µCi
of [
-
P]ATP. After 10 min at 37 °C, the
reaction was stopped by the addition of 1 ml of lysis buffer containing
20 mM EDTA.
P-Labeled proteins were solubilized
in SDS sample buffer prior to separation by SDS-polyacrylamide gel
electrophoresis and were visualized by autoradiography at
70
°C.
RESULTS
Identification of in Vitro Lipid Kinase Activity
Immunoprecipitated by p85 mAb
The p85 immunoprecipitates were
assayed for lipid kinase activity, and the production of
[P]PtdIns was shown to be linear for at least 30
min under the conditions described under ``Experimental
Procedures'' (Fig. 2A). The lipid kinase activity
present in p85 mAb immunoprecipitates (Fig. 2B, lane2) was also inhibited by the reported PI 3-kinase
inhibitor wortmannin (Fig. 2A, lane3). The lipid kinase activity immunoprecipitated by p85
(Fig. 2B, lane 2) was further confirmed as PI
3-kinase by HPLC analysis of glycerophosphorylinositol derivatives of
the [
P]PtdInsP formed (Fig. 2C)
and by comparison with known standards. In contrast, HPLC analysis of
glycerophosphorylinositol derivatives formed following incubation of
total cell lysates with PtdIns under identical conditions
(Fig. 2B, lane1) revealed predominant
formation of [
P]PtdIns(4)P
(Fig. 2D). The apparent K
for PtdIns is 15.7
5.7 µM (n = 7), which was determined by varying the concentration of
PtdIns (keeping the molar ratio of phosphatidylinositol to
phosphatidylserine at 1).
Figure 2:
Immunoprecipitation of PI 3-kinase
activity by p85 mAb. Jurkat cells (2 10
) were
lysed, and all lysates were subjected to immunoprecipitation with a p85
mAb. A, p85 immunoprecipitates incubated with PtdIns and
analyzed for PI 3-kinase activity at the times shown. B, p85
immunoprecipitates incubated with PtdIns in the absence (lane2) or presence (lane3) of 100
nM wortmannin. The washed p85 immunoprecipitates and 40 µl
of total resting Jurkat cell lysate (2
10
cells;
lane1) were analyzed for PI 3-kinase activity.
Extraction and thin-layer chromatographic separation of the lipid
products were performed as described under ``Experimental
Procedures.'' C, HPLC elution profile of the
glycerophosphorylinositol derivatives of
[
P]PtdIns(3)P (GroPIns(3)P) formed by
p85 immunoprecipitates following incubation with PtdIns in B (lane 2). D, HPLC elution profile of the
glycerophosphorylinositol derivatives of
[
P]PtdIns(4)P (GroPIns(4)P) formed by
Jurkat lysates in B (lane
1).
Effect of L-chiro-Ins(2,3,5)P
L-chiro-Ins(2,3,5)Pand L-chiro-Ins(2,3,5)PS
on PI 3-Kinase
Activity
(Fig. 1, compound 2) and
L-chiro-Ins(2,3,5)PS
(compound 3) induced concentration-dependent inhibition of the PI 3-kinase
activity present in p85 mAb immunoprecipitates with IC
values of 5
3.5 and 20
5 µM,
respectively (calculated using a substrate concentration of 100
µM PtdIns) (). The inhibition by
L-chiro-Ins(2,3,5)P
(Fig. 3) and
L-chiro-Ins(2,3,5)PS
(data not shown) was
reversible (Fig. 3), with apparent K
values of 39
14 and 76
34 µM,
respectively ().
L-chiro-Ins(2,3,5)P
( Fig. 4and
) and L-chiro-Ins(2,3,5)PS
() had no effect on the K
for immunoprecipitated PI 3-kinase, while V
was markedly decreased, indicating noncompetitive inhibition of
PI 3-kinase by these inositol phosphate analogues. The percentage
inhibition of PI 3-kinase activity induced by these compounds was
similar after preincubation times ranging from 1 to 30 min (data not
shown).
Figure 3:
Effect of
L-chiro-Ins(2,3,5)P on in vitro PI 3-kinase activity. Jurkat cells (2
10
) were
sedimented alone, and all lysates were subjected to immunoprecipitation
with a p85 mAb. A, the washed immunoprecipitates were analyzed
for PI 3-kinase activity after a 15-min pretreatment with vehicle or
L-chiro-Ins(2,3,5)P
(30 µM)
using PtdIns as a substrate at the concentrations indicated. In the
middlepanel, the immunoprecipitates were washed free
of L-chiro-Ins(2,3,5)P
before the
reaction was initiated. Extraction and thin-layer chromatographic
separation of the lipid products (A and B) were
performed as described under ``Experimental Procedures.''
Phospholipids were visualized by autoradiography at
70 °C.
B, PtdIns(3)P was formed following incubation of p85 with
increasing concentrations of PtdIns (0.5-100 µM)
after a 15-min pretreatment with vehicle (
), 100 µML-chiro-Ins(2,3,5)P
(
), or 100
µML-chiro-Ins(2,3,5)P
followed by washing three times in lipid kinase assay buffer
(
). Data shown are from a single preparation representative of at
least five others.
Figure 4:
Effect of
L-chiro-Ins(2,3,5)P on K and
V
for PI 3-kinase with respect to PtdIns.
A, Jurkat cells (2
10
) were sedimented
alone, and all lysates were subjected to immunoprecipitation with a p85
mAb. The washed immunoprecipitates were analyzed for PI 3-kinase
activity after a 15-min pretreatment with vehicle or
L-chiro-Ins(2,3,5)P
using PtdIns as a
substrate at the concentrations indicated. Extraction and thin-layer
chromatographic separation of the lipid products (A-C)
were performed as described under ``Experimental
Procedures.'' Phospholipids were visualized by autoradiography at
70 °C. B, PtdIns(3)P was formed following
incubation of p85 with increasing concentrations of PtdIns (1-200
µM) after a 15-min pretreatment with vehicle (
) or
30 µM (
) or 100 µM (
)
L-chiro-Ins(2,3,5)P
. C, the
results in B are presented as double-reciprocal plots for the
substrate PtdIns in the presence of vehicle (
) or 30
µM (
) or 100 µM (
)
L-chiro-Ins(2,3,5)P
. Data shown are from
a single preparation representative of at least five
others.
Effect of Other Inositol Phosphate Analogues on PI
3-Kinase Activity
The PI 3-kinase activity present in p85
immunoprecipitates was not affected by
D-Ins(1,4,5)P, L-Ins(1,4,5)P
,
or the major Ins(1,4,5)P
metabolites Ins(1,3,4,5)P
() and D- and
L-myo-inositol 1,3,4-trisphosphate. A number of other
synthetic analogues, including DL-Ins(1,4,6)P
,
L-chiro-inositol 1,4,6-trisphosphorothioate
(L-chiro-Ins(1,4,6)PS
),
L-myo-inositol 1,4,5-trisphosphorothioate
(L-Ins(1,4,5)PS
), and myo-inositol
1,3,5-trisphosphorothioate (Ins(1,3,5)PS
) () at
concentrations of 100 µM, also did not inhibit PI 3-kinase
activity (see ``Discussion'').
Synthesis of Benzene 1,2,4-Trisphosphate and Effect on PI
3-Kinase Activity
Benzene 1,2,4-trisphosphate (Fig. 1,
compound 6) was synthesized by a markedly improved method to
that previously reported
(26) , involving isolation and full
spectral and analytical characterization of an intermediate
1,2,4-tris(diethylphospho)benzene, followed by deprotection, rigorous
purification of the anionic product, and full spectral
characterization. This very loosely related structural analogue of
Ins(1,4,5)P was found to inhibit PI 3-kinase activity in
p85 mAb immunoprecipitates with an IC
value of 25
2.5 µM (n = 5) () when using
100 µM PtdIns as a substrate. This inhibition of PI
3-kinase by Bz(1,2,4)P
was reversible (Fig. 5).
Bz(1,2,4)P
( Fig. 6) had no effect on
the K
for immunoprecipitated PI 3-kinase,
while V
was markedly decreased, indicating
noncompetitive inhibition of PI 3-kinase with an apparent
K
of 93.6
15 µM.
Figure 5:
Inhibition of in vitro PI
3-kinase activity by Bz(1,2,4)P. Jurkat cells (2
10
) were sedimented alone, and all lysates were subjected
to immunoprecipitation with a p85 mAb. A, the washed
immunoprecipitates were analyzed for PI 3-kinase activity after a
15-min pretreatment with vehicle or Bz(1,2,4)P
(100
µM) using PtdIns as a substrate at the concentrations
indicated. In the middlepanel, the
immunoprecipitates were washed free of Bz(1,2,4)P
before
the reaction was initiated. Extraction and thin-layer chromatographic
separation of the lipid products were performed as described under
``Experimental Procedures.'' Phospholipids were visualized by
autoradiography at
70 °C. B, PtdIns(3)P was formed
following incubation of p85 with increasing concentrations of PtdIns
(0.5-100 µM) after a 15-min pretreatment with
vehicle (
), 100 µM Bz(1,2,4)P
(
),
or 100 µM Bz(1,2,4)P
followed by washing three
times in lipid kinase assay buffer (
). Data shown are from a
single preparation representative of at least five
others.
Figure 6:
Effect of Bz(1,2,4)P3 on K and
V values for PI 3-kinase with respect to PtdIns.
A, Jurkat cells (2
10
) were sedimented
alone, and all lysates were subjected to immunoprecipitation with a p85
mAb. The washed immunoprecipitates were analyzed for PI 3-kinase
activity after a 15-min pretreatment with vehicle or Bz(1,2,4)P
using PtdIns as a substrate at the concentrations indicated.
Extraction and thin-layer chromatographic separation of the lipid
products (A-C) were performed as described under
``Experimental Procedures.'' Phospholipids were visualized by
autoradiography at
70 °C. B, PtdIns(3)P was formed
following incubation of p85 with increasing concentrations of PtdIns
(0.5-100 µM) after a 15-min pretreatment with
vehicle (
) or 10 µM (
) or 100 µM
(
) Bz(1,2,4)P
. C, the results in B are presented as double-reciprocal plots for the substrate PtdIns
in the presence of vehicle (
) or 100 µM
Bz(1,2,4)P
(
). Data shown are from a single
preparation representative of at least five
others.
Effect of
L-chiro-Ins(2,3,5)P
The
specificity of the inhibition of PI 3-kinase by
L-chiro-Ins(2,3,5)P,
L-chiro-Ins(2,3,5)PS
, and
Bz(1,2,4)P
on PI 4-Kinase Activity
,
L-chiro-Ins(2,3,5)PS
, and
Bz(1,2,4)P
was determined by analyzing the effects of these
compounds on PtdIns 4-kinase.
L-chiro-Ins(2,3,5)P
,
L-chiro-Ins(2,3,5)PS
, and
Bz(1,2,4)P
at concentrations of 100 µM had no
effect on the predominantly PtdIns 4-kinase activity present in T cell
lysates (data not shown). The lack of effect of these compounds with
respect to the inhibition of kinase activity in lysates may be due to
instability of these analogues in cell lysates. Previous work has shown
that PtdIns 4-kinase binds to protein A
(34) . Hence, protein
A-Sepharose beads that had been previously incubated with post-nuclear
lysates at 4 °C contained PtdIns 4-kinase activity as determined by
HPLC analysis of glycerophosphorylinositol derivatives formed during
in vitro lipid kinase experiments (data not shown).
L-chiro-Ins(2,3,5)P
and Bz(1,2,4)P
at concentrations of 100 µM had no effect on the
PtdIns 4-kinase activity associated with protein A (Fig. 7).
Figure 7:
Effect of
L-chiro-Ins(2,3,5)P and
Bz(1,2,4)P
on PI 4-kinase activity. Jurkat cells (2
10
) were sedimented alone, and all lysates were subjected
to immunoprecipitation with protein A-Sepharose. The washed
immunoprecipitates were analyzed for PtdIns kinase activity after a
15-min pretreatment with vehicle (
), 100 µML-chiro Ins(2,3,5)P
(
), or 100
µM Bz(1,2,4)P
(
) using PtdIns as a
substrate. Extraction and thin-layer chromatographic separation of the
lipid products were performed as described under ``Experimental
Procedures.'' Phospholipids were visualized by autoradiography at
70 °C. Data shown are from a single preparation
representative of at least five others.
Effect of
L-chiro-Ins(2,3,5)P
L-chiro-Ins(2,3,5)P,
L-chiro-Ins(2,3,5)PS
, and
Bz(1,2,4)P
on in Vitro Protein Kinase
Activity
(Fig. 8),
L-chiro-Ins(2,3,5)PS
, and
Bz(1,2,4)P
(data not shown) had no effect on the activity
of a coprecipitated protein-serine kinase that is known to
phosphorylate the p85 subunit
(31, 35, 36) .
Figure 8:
Effect of
L-chiro-Ins(2,3,5)P on in vitro protein kinase activity coassociated with p85 immunoprecipitates.
Jurkat cells (2
10
) were sedimented alone, and all
lysates were subjected to immunoprecipitation with a p85 mAb. In
vitro protein kinase reactions were performed on these washed p85
immunoprecipitates as described under ``Experimental
Procedures'' in the presence of
L-chiro-Ins(2,3,5)P
(3-100
µM). The immunoprecipitates were washed, and proteins were
resolved by SDS-polyacrylamide gel electrophoresis and visualized by
autoradiography at
70 °C. Migration of the molecular mass
standards is indicated to the left.
DISCUSSION
Synthetic routes have been developed (reviewed in Ref. 37)
that have enabled the preparation of natural and unnatural inositol
phosphates and their structurally modified analogues. This approach has
yielded inositol phosphate analogues that have been shown to act as
inhibitors of enzymes controlling the metabolism of
Ins(1,4,5)P, particularly inositol-phosphate 5-phosphatase
and Ins(1,4,5)P
3-kinase
(23) . In this study, we
have immunoprecipitated PI 3-kinase activity using a mAb to the
regulatory p85 subunit, which is known to coassociate with the
catalytic p110 subunit
(1, 2, 38) . We have also
demonstrated the inhibition of leukemic T cell-derived PI 3-kinase by
two synthetic inositol phosphate analogues,
L-chiro-Ins(2,3,5)P
(Fig. 1,
compound 2) and L-chiro-Ins(2,3,5)PS
(compound 3), and by a loosely related Ins(1,4,5)P
structural analogue, Bz(1,2,4)P
(compound 6).
These compounds did not affect the lipid kinase activity present in
total cell lysates, which was predominantly PtdIns 4-kinase, or a
serine kinase activity
(31, 35, 36) coprecipitated with p85 immunoprecipitates.
L-chiro-Ins(2,3,5)P
has previously been
reported to mobilize intracellular calcium, albeit less potently than
Ins(1,4,5)P
(23, 39, 40, 41) ,
and inhibits Ins(1,4,5)P
5-phosphatase
(23, 40) . Its phosphorothioate
derivative, L-chiro-Ins(2,3,5)PS
, is a
partial agonist for calcium release
(25, 39, 41) and a more potent inhibitor of 5-phosphatase
(23) .
Both analogues also inhibit Ins(1,4,5)P
3-kinase with a
similar affinity and are to date the most effective inositol phosphate
analogue inhibitors of this enzyme
(23) . Bz(1,2,4)P
,
which has the inositol ring replaced by a flat benzene ring but
possesses three phosphate groups in a similar spatial arrangement to
that of Ins(1,4,5)P
, inhibits Ins(1,4,5)P
5-phosphatase and Ins(1,4,5)P
3-kinase less
effectively than L-chiro-Ins(2,3,5)P
or
L-chiro-Ins(2,3,5)PS
and does not
mobilize intracellular calcium
(26) .
(23, 42) ,
L-Ins(1,4,5)PS
(23, 43, 44) ,
Ins(1,3,5)PS
(23, 43, 44) , and
L-chiro-Ins(1,4,6)PS
(23, 45) ,
all of which were found not to inhibit PI 3-kinase at a concentration
of 100 µM ( and data not shown). Hence, the
inhibition of PI 3-kinase action appears not to be a general feature of
analogues that inhibit 5-phosphatase. In addition, several other
naturally occurring and synthetic inositol polyphosphate analogues were
examined for their ability to inhibit PI 3-kinase activity. These
compounds included
DL-3-O-methyl-myo-inositol
1,4,5-trisphosphate
(46) , scyllo-inositol
1,2,4,5-tetrakisphosphate
(47) ,
DL-scyllo-inositol 1,2,4-trisphosphate
(DL-scyllo-Ins(1,2,4)P
) (47, 48),
DL-myo-inositol
1,2,4,5-tetrakisphosphate
(47, 49) ,
2,5-di-O-methyl-myo-inositol
1,3,4,6-tetrakisphosphate
(23) , D- and
L-myo-inositol 1,3,4-trisphosphate (50), and
DL-Ins(1,4,6)P
(28) , which were all found
not to inhibit PI 3-kinase at 100 µM (data not shown).
(Fig. 1,
compound 2)
(23, 39, 40, 41) resembles Ins(1,4,5)P
(compound 1) in
all respects except one: the equatorial 3-hydroxyl group of
Ins(1,4,5)P
is replaced by an axial hydroxyl group at the
L-chiro-Ins(2,3,5)P
1-position. In
addition, the analogue D-Ins(1,4,6)P
(Fig. 1, compound 4)
(28) , in its expected
receptor binding conformation relative to Ins(1,4,5)P
, also
resembles L-chiro-Ins(2,3,5)P
except that
the axial 6-hydroxyl group of the latter, which is equivalently located
at the 2-position of Ins(1,4,5)P
, is replaced by an
equatorial 3-hydroxyl group in D-Ins(1,4,6)P
.
These subtle modifications reduce the potency of both compounds with
respect to calcium mobilization in comparison with Ins(1,4,5)P
since both L-chiro-Ins(2,3,5)P
and
DL-Ins(1,4,6)P
are less potent than
Ins(1,4,5)P
(23, 28, 40) .
scyllo-Ins(1,2,4)P
(Fig. 1, compound 5) is identical in structure to Ins(1,4,5)P
except
that it possesses an equatorial 5-hydroxyl group and is equivalent to
the 2-position in Ins(1,4,5)P
, which has an axial hydroxyl
group. scyllo-Ins(1,2,4)P
is only marginally less
potent than Ins(1,4,5)P
with respect to calcium
mobilization
(47) and was inactive as a PI 3-kinase inhibitor.
The ability of inositol phosphate analogues to inhibit PI 3-kinase thus
appears to be very sensitive to orientations of hydroxyl groups of the
inositol ring since Ins(1,4,5)P
, Ins(1,4,6)P
,
and scyllo-Ins(1,2,4)P
, which all resemble
L-chiro-Ins(2,3,5)P
except for
orientation of the equivalent 2- and 3-position hydroxyl groups
(Ins(1,4,5)P
numbering), do not inhibit PI 3-kinase.
Moreover, alternative modification at the 3-position of
Ins(1,4,5)P
only, as in
3-O-Me-Ins(1,4,5)P
, produced an inactive compound
with respect to PI 3-kinase inhibition.
numbering) for
inositol phosphate analogues to exhibit the ability to inhibit PI
3-kinase activity. It is not surprising that a structural perturbation
at the equivalent myo-inositol D-3-position, the site
of phosphorylation by PI 3-kinase, results in inhibitor generation.
Interestingly, the orientation of hydroxyl groups of the inositol ring
has also been reported to determine recognition of Ins(1,4,5)P
by its receptor and the metabolic enzymes Ins(1,4,5)P
5-phosphatase and Ins(1,4,5)P
3-kinase
(37, 51) . Hence, it appears from the data
presented in this study that modification of hydroxyl group orientation
at the crucial 3-position can also confer PI 3-kinase inhibitory
properties upon certain inositol phosphate analogues.
,
L-chiro-Ins(2,3,5)PS
, and
Bz(1,2,4)P
is reversible is not surprising since the
inhibitory action of L-chiro-Ins(2,3,5)P
on Ins(1,4,5)P
3-kinase is of a reversible
competitive nature (23, 24). Indeed, these compounds were used in this
study on the basis that they may compete with the lipid substrate for
the active site on the PI 3-kinase. The data shown here suggest that
the inhibition of PI 3-kinase induced by
L-chiro-Ins(2,3,5)P
and its
phosphorothioate derivative as well as Bz(1,2,4)P
is
noncompetitive and may involve mechanisms other than competition with
the lipid substrate at the active site of the enzyme. There are several
explanations for these observations. First, these inositol phosphate
analogues may modulate enzyme activity by interacting with PI 3-kinase
at a site other than the active site. This may be an important
regulatory site present on either the p85 or p110 subunit and a
potential target site for future inhibitor development. The possible
existence of such a regulatory site and its endogenous ligand (and even
whether such a ligand is an inositol phosphate) remains to be
established since the most obvious candidates for binding to this site
are Ins(1,4,5)P
and Ins(1,3,4,5)P
, which have
been shown to have no effect on PI 3-kinase activity. Second,
L-chiro-Ins(2,3,5)P
and Bz(1,2,4)P
may differ sufficiently in structure from the numerous inactive
inositol phosphates we have studied in that they are recognized by PI
3-kinase and are able to compete with the substrate lipid for access to
the active site. However, these inositol phosphate analogues lack the
glycerol backbone and acyl groups of the natural substrate lipids that
may mediate or facilitate optimum enzyme-substrate interaction. In
these circumstances, it is possible to envisage the PI 3-kinase
inhibitors binding to both enzyme and enzyme-lipid substrate complexes
and thus fulfilling the criteria for noncompetitive
inhibition
(52) . Third, the apparent K
values for inhibition of PI 3-kinase by
L-chiro-Ins(2,3,5)P
,
L-chiro-Ins(2,3,5)PS
, and in particular
Bz(1,2,4)P
are substantially higher () than the
IC
values. This discrepancy may imply mixed competitive
and noncompetitive inhibition, which can be envisaged given the
proposed interactions described above. The data in show
that the calculated K
value for PI
3-kinase has an error of
30%, while the data in show
that the calculated K
values have errors
of
15-30%. The apparent failure to detect an increased
K
for PI 3-kinase in the presence of the
inhibitory inositol phosphate analogues that would be expected for
mixed competitive and noncompetitive inhibition
(52) may be
obscured by the relatively large error margins associated with these
experiments. The apparent K
values for
inhibition of PI 3-kinase by
L-chiro-Ins(2,3,5)P
,
L-chiro-Ins(2,3,5)PS
, and in particular
Bz(1,2,4)P
are substantially higher () than the
reported K
values for inhibition of
Ins(1,4,5)P
3-kinase. It should be noted from ,
however, that the K
for Ins(1,4,5)P
3-kinase is much lower than the K
for PI 3-kinase. While it is not certain precisely how these
inhibitors interact with PI 3-kinase, one possibility is that their
inhibitory action may be due to nonselective inhibition of the transfer
of phosphate from ATP. However, this is unlikely since the analogues
had no effect on the serine phosphorylation of p85 mediated by a
tightly associated serine kinase (31, 35, 36).
,
L-chiro-Ins(2,3,5)PS
, and
Bz(1,2,4)P
inhibit both Ins(1,4,5)P
5-phosphatase and Ins(1,4,5)P
3-kinase with varying
potency
(23, 26, 40) and have been suggested to
be important lead compounds in the development of specific small
molecule inhibitors of these enzymes. At present, no compound has been
identified as a PI 3-kinase inhibitor that is not also an
Ins(1,4,5)P
5-phosphatase or 3-kinase inhibitor.
Bz(1,2,4)P
represents an especially important lead since
chemical modification of this compound is much easier than are the
often extensive synthetic routes for preparation of inositol phosphate
analogues. We have developed a much improved and high yielding
synthetic route to Bz(1,2,4)P
than that previously reported
by other workers (26), who neither purified nor fully characterized
their intermediate and final products.
3-kinase.
Furthermore, the analogues described in this study are highly polar,
and these agents therefore do not permeate the cell membrane. Thus,
while they can be considered useful agents in cell-free or
permeabilized cell preparations, this does not apply when considering
D-3-phosphatidylinositol lipid generation in intact cells,
where it will be necessary to make analogues with further refinements
and developments to generate effective apolar membrane-permeable
inhibitors. Nevertheless, the identification of inositol phosphate
analogues that act as PI 3-kinase inhibitors and that presumably
compete with the phosphatidylinositols for substrate recognition by PI
3-kinase provides novel key pharmacological tools for intervention at a
crucial component of this postulated signaling pathway.
Table: Characteristics of the inhibition of
Ins(1,4,5)P5-phosphatase,
Ins(1,4,5)P
3-kinase, and PI 3-kinase by
inositol phosphate analogues and Bz(1,2,4)P
values for Ins(1,4,5)P
and Ins(1,4,5)P
3-kinase are representative and may
vary according to source of enzyme and individual investigators.
Table: Kand
V
values for immunoprecipitated PI
3-kinase
and
V
values vary
2-4-fold from
preparation to preparation. Data for each analogue are derived from
separate preparations. Mean K
values
(
S.E.) for immunoprecipitated PI 3-kinase with respect to
PtdIns are given in parentheses and are derived from seven separate
preparations.
,
D-myo-inositol 1,4,5-trisphosphate;
DL-Ins(1,4,6)P
, DL-myo-inositol
1,4,6-trisphosphate;
L-chiro-Ins(1,4,6)PS
,
L-chiro-inositol 1,4,6-trisphosphorothioate;
Ins(1,3,4,5)P
, D-myo-inositol
1,3,4,5-tetrakisphosphate; Ins(1,3,5)PS
,
myo-inositol 1,3,5-trisphosphorothioate;
L-chiro-Ins(2,3,5)P
,
L-chiro-inositol 2,3,5-trisphosphate;
L-chiro-Ins(2,3,5)PS
,
L-chiro-inositol 2,3,5-trisphosphorothioate;
L-Ins(1,4,5)PS
, L-myo-inositol
1,4,5-trisphosphorothioate;
DL-scyllo-Ins(1,2,4)P
,
DL-scyllo-inositol 1,2,4-trisphosphate;
Bz(1,2,4)P
, benzene 1,2,4-trisphosphate; mAb, monoclonal
antibody; HPLC, high pressure liquid chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.