L-aminoacyl-triazine derivatives are isoform-selective PI3Kβ inhibitors that target nonconserved Asp862 of PI3Kβ

Article abstract of DOI:10.1021/ml300336j

A series of aminoacyl-triazine derivatives based upon the pan-PI3K inhibitor ZSTK474 were identified as potent and isoform-selective inhibitors of PI3Kβ. The compounds showed selectivity based upon stereochemistry with l-amino acyl derivatives preferring PI3Kβ, while their d-congeners favored PI3Kδ. The mechanistic basis of this inhibition was studied using site-directed mutants. One Asp residue, D862, was identified as a critical participant in binding to the PI3Kβ-selective inhibitors, distinguishing this class from other reported PI3Kβ-selective inhibitors. The compounds show strong inhibition of cellular Akt phosphorylation and growth of PTEN-deficient MD-MBA-468 cells.

Full text of DOI:10.1021/ml300336j

Letter
pubs.acs.org/acsmedchemlett
L‑Aminoacyl-triazine Derivatives Are Isoform-Selective PI3Kβ
Inhibitors That Target Nonconserved Asp862 of PI3Kβ
Jo-Anne Pinson, Zhaohua Zheng, Michelle S. Miller, David K. Chalmers, Ian G. Jennings,
and Philip E. Thompson*
Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052 Australia.
S
* Supporting Information
ABSTRACT: A series of aminoacyl-triazine derivatives based upon the pan-PI3K inhibitor
ZSTK474 were identified as potent and isoform-selective inhibitors of PI3Kβ. The compounds
showed selectivity based upon stereochemistry with L-amino acyl derivatives preferring PI3Kβ,
while their D-congeners favored PI3Kδ. The mechanistic basis of this inhibition was studied using
site-directed mutants. One Asp residue, D862, was identified as a critical participant in binding to
the PI3Kβ-selective inhibitors, distinguishing this class from other reported PI3Kβ-selective
inhibitors. The compounds show strong inhibition of cellular Akt phosphorylation and growth of
PTEN-deficient MD-MBA-468 cells.
KEYWORDS: PI3 kinase, p110β, ZSTK474, cancer
he phosphatidylinositol 3-kinases (PI3K) are a family of
Tlipid kinases that regulate intracellular signaling for
numerous cellular events such as cell migration, growth, and
survival.1 Because of their important roles in signal trans-
duction, dysregulation in the PI3K pathway could lead to
various diseases including cancer.2 Two of the key genetic
alterations identified for this pathway in cancer are mutation in
the PIK3CA gene, which encodes p110α (PI3Kα catalytic
subunit),3 and the loss of the tumor suppressor, phosphatase
and tensin homologue (PTEN).4 Recently, there has been
accumulating evidence showing that PI3Kβ plays a key role in
tumorigenesis driven by PTEN loss.5 It was found that down-
regulation of the PIK3CB gene, which encodes p110β (PI3Kβ
catalytic subunit), inhibited PI3K signaling as well as growth
both in vitro and in vivo. In an animal prostate cancer model,
the ablation of p110β blocked tumorigenesis and decreased Akt
Figure 1. Nonconserved regions 1 and 2 of the PI3Kγ binding site
(shown in space-filling representations). PDB code: 1E8X.
phosphorylation, but the same results were not observed by
p110α ablation.6
The majority of inhibitors currently in clinical trials are class I
pan-PI3K inhibitors.7 Because each of the PI3K isoforms has
their own although overlapping physiological roles, isoform-
selective PI3K inhibitors may hold some therapeutic advantages
with respect to reducing off-target effects. In recent years,
isoform-selective inhibitors have emerged, and compounds
855 to 862 encompasses a loop that sits under the ribose
pocket and influences the binding of PI3Kα selective inhibitor
A-66 (a BYL719 analogue).11 Region 2 from PI3Kβ 772−788
corresponds to the protein kinase “P-loop”.11 This region has
been shown to dictate selective inhibitor binding in a number
of waysfirst, a conserved methionine residue can shift to
expose a cryptic binding pocket, as in the so-called “propeller-
shaped” mode of binding of PI3Kδ-selective compounds like
PIK-39 (a CAL-101 analogue).12 Other PI3Kδ-selective
inhibitors have been shown to access a “tryptophan shelf”
adjacent to a nonconserved threonine. Also, the PI3Kα-
selective inhibitors PIK75 and J-32 have been shown to be
such as BYL719, a PI3Kα selective inhibitor, and CAL-101,8,9
a
PI3Kδ-specific inhibitor, are now in phase I/II clinical trials.
While these and other isoform-selective inhibitors have been
reported, there is still relatively scant understanding of the
mechanisms underpinning selectivity. From the structure of the
adenosine triphosphate (ATP)-bound state of the enzyme
(PDB: 1E8X), it is apparent that much of the inner core of the
binding site is highly conserved across the class 1 isoforms.10
However, two nonconserved regions of the binding site have
also been identified as capable of executing selective
interactions with inhibitors (Figure 1). Region 1 from PI3Kβ
Received: October 14, 2012
Accepted: December 20, 2012
Published: December 20, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
sensitive to mutation at nonconserved residues of region 2.11
There are clearly other mechanisms by which inhibitors exhibit
isoform selectivity. This is especially apparent in PI3Kγ-
selective inhibitors that show interaction only with conserved
residues of the binding site and induce little conformational
change from the ATP-bound state.13
Until recently, the pharmacological evaluation of PI3Kβ
inhibition has rested upon a series of compounds described by
Thrombogenix/Kinacia.14,15 These included TGX221 and
KN309 (Figure 2). Astra-Zeneca has subsequently progressed
accommodated in region 2, via the “propeller-shaped” mode
described above. We had previously ruled out an interaction
with the region 1 residues as the basis for selectivity.17
Recently, new PI3Kβ-selective compounds reminiscent of
TGX221 have been reported. GSK2636771 has progressed to
phase I/IIa clinical trials to treat advanced solid tumors with
PTEN deficiency. Other analogous series have been
described,18−23 and cocrystals of compounds with PI3Kγ
(PDB: 4FJY, 4FJZ, and 4G11) and PI3Kδ (4AJW) support
the analogy between these and the “propeller-shaped”
compounds. The only reported X-ray structure of PI3Kβ in
complex with the pan-PI3K inhibitor GDC9041 (PDB: 2Y3A)
displays a conventional “flat” binding pose.24
Our hypothesis was that isoform-selective inhibition of
PI3Kβ should be achievable by targeting the nonconserved
residues of region 1. In particular, the acidic Asp862 residue at
PI3Kβ is exchanged for Gln, Lys, and Asn in PI3Kα, -γ, and -δ,
respectively. Adaptation of the pan-PI3K inhibitor ZSTK474
(Figure 2),25 which projects one morpholinyl ring toward this
region, was considered the logical template compound.12 We
have identified compounds that selectively inhibit PI3Kβ and
moreover have demonstrated the role of D862 by showing that
mutagenesis of that residue prevents inhibition by these
compounds.
The synthesis of the substituted triazines was performed
largely as previously reported (see the Supporting Informa-
tion).26−28 Condensation of protected amino acid derivatives to
piperazinyl analogues of ZSTK474 yielded the N-protected
derivatives, which could be deprotected to give the free
aminoacyl compounds 17 and 19−28. Alternatively condensa-
tion with N-acetylglycine or N,N-dimethylglycine gave the
products 18 and 29, respectively.
Figure 2. Structures of PI3K inhibitors.
the optically pure R-enantiomer of KN309 into human trials as
AZD6482 (aka KIN193).16 The molecular basis for the
observed selectivity has not yet been established, although
Knight et al. proposed that TGX280 (PIK108) could be
The PI3K assay data are summarized in Table 1 and identify
that the substituent has a significant influence upon isoform
selectivity, consistent with our hypothesis. Compound 17
Table 1. Structure and Inhibition of PI3K Isoforms by ZSTK474 Analogues
a
PI3K isoform IC50 (nM)
compd
AA residue
Gly
α
β
γ
δ
β/δ selectivity ratio
ZSTK474
17
6
6
35
54
31
38
3
0.5
3.0
1500
42
9900
380
110
26
18
Ac-Gly
L-Ala
0.48
15.7
0.070
34.8
<0.36
15.6
5.4
19
3500
3700
4700
>10 μM
6300
3300
2200
2300
720
3600
490
70
20
D-Ala
1000
63
2000
21
L-Phe
>100 μM
>10 μM
>10 μM
>10 μM
>10 μM
>10 μM
3900
2200
3600
1000
1200
390
670
58
22
D-Phe
>10 μM
67
23
L-Ile
24
L-Tyr
220
25
L-Pro
26
15.1
13.4
0.6
26
L-Lys
50
27
β-Ala
98
28
N-Me-L-Ala
N,N-diMeGly
1500
1600
260
8000
290
330
1.1
29
2400
>10 μM
0.14
a
IC50 values are means of at least two duplicate experiments (Kinase-Glo). Standard errors were all within 25% of the mean. Assays were performed
using 10 μM ATP.
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showed potent dual inhibition of PI3Kβ and PI3Kδ with very
poor inhibition of the PI3Kα and PI3Kγ isoforms. Incorpo-
ration of an N-acetylglycine residue 18 resulted in pan-PI3K
potency, suggesting the importance of the amino function to
the observed selectivity.
restricts the interaction to D862 alone and blocks possible
interaction with E858, which is also an acidic residue in PI3Kδ
(D831). This may also explain the potency of 17 and 20 at
PI3Kδ.
Docking simulations support this model. In the wild-type
enzyme, 21 adopted the anticipated pose closely overlaying the
reported pose of ZSTK474 in PI3Kδ12 and making a specific
interaction of the free amine with D862 (Figure 3). In the
Compounds 19−29 were all PI3Kβ and/or PI3Kδ preferring
ligands. Strikingly, the introduction of an L-alanine substituent
resulted in a compound 19 with 15-fold selectivity for PI3Kβ
over PI3Kδ, while the opposite stereoisomer with a D-alanine
substituent 21 showed 15-fold selectivity for PI3Kδ over
PI3Kβ. Pursuing the potential for L-amino acids providing for
PI3Kβ selectivity in general held true. Most selective was L-Phe
(21), which showed 35-fold selectivity for PI3Kβ over PI3Kδ
with the L-Ile (23), L-Tyr (24), L-Pro (25), and L-Lys (26)
derivatives showing 6−16-fold selectivity. Homologation to β-
Ala (27) and further substitution on the amino group as in 28
and 29 dropped the preference for PI3Kβ significantly.
To test the premise that the observed PI3Kβ selectivity was
due to the interaction between the primary amino group of
inhibitors and the nonconserved residues of the binding site,
compounds were tested against a variety of mutant forms of
PI3Kβ where the nonconserved residues were exchanged for
their equivalent residues in PI3Kα.
The PI3Kβ-selective compounds were very sensitive to
mutation at region 1 residue D862Q (see Supplementary
Figure 1 in the Supporting Information). The IC50 values were
determined for 19, 21, and 23 against the WT and mutant
D862Q form as well as the WT and reciprocal mutant in PI3Kα
(Table 2). Note that the mutations do not hinder the catalytic
Figure 3. Docking solution for compound 21 into a model of PI3Kβ
derived from crystal structure PDB: 2Y3A.
D862Q mutant form, not only is this specific interaction lost,
but the compound can no longer adopt this pose, suggesting
that the aspartyl residue acts to compensate for other
unfavorable interactions associated with the inclusion of a
phenylalanine substituent.
Table 2. Inhibition of PI3K Isoforms and in Vitro Mutants
by ZSTK474 Analogues
Finally, we assessed whether these compounds exhibited cell-
based activity that might make them worthy of further
pharmacological study and comparison to other recently
reported PI3Kβ inhibitors.19 The PTEN-deficient cell line
MDA-MB-468 was used to measure inhibition of Akt
phosphorylation and inhibition of cell growth. Dose-dependent
inhibition of both activities was observed with each of the
inhibitors tested. Compound 21 showed strong cellular
inhibition of Akt phosphorylation relative to the direct enzyme
assay with IC50 values <10 nM (Supplementary Figure 2 in the
Supporting Information). Compound 21 showed comparable
potency to ZSTK474 in cell growth despite both lower potency
at PI3Kβ and poor potency at the other isoforms (Table 3).
a
PI3K isoform IC50 (nM)
fold change fold change
αWT→
αQ859D
PI3Kα PI3Kα PI3Kβ PI3Kβ
βWT→
compd
WT
Q859D
WT
D862Q
βD862Q
19
21
23
8200
12000
9200
2300
8100
2800
62
74
890
6000
1400
↓3.5
↓1.4
↓3.3
↑14
↑82
↑10
140
a
IC50 values are means of at least two duplicate experiments (Kinase-
Glo). Assays were performed using 100 μM ATP. A full table including
standard errors is provided as Supplementary Table 2 in the
Supporting Information.
properties of the mutant enzymes (Supplementary Table 1 in
the Supporting Information). Each of the compounds shows a
greater than 10-fold change in IC50 against the mutant form of
PI3Kβ. The IC50 of 21 drops 82-fold as compared to the wild-
type isoform upon introduction of the D862Q mutation. The
reciprocal mutation in PI3Kα D859Q does not restore
inhibitory potency of 21 at PI3Kα, although some enhance-
ment in potency is observed for compounds 19 and 23. These
data clearly distinguish the mechanism of selectivity of these
compounds from that of TGX221 and TGX286, which are
unaffected by the mutation D862Q in PI3Kβ.17
Table 3. Inhibition of Cell Growth in Breast Cancer Cell
Line by ZSTK474 Analogues
MDA-MB-468 growth EC50 (μM)
ZSTK474
3.2 1.4
15 5.3
13 1.2
4.6 1.1
11 1.1
17
19
21
23
The data from these studies suggest a clear and dramatic
influence of aminoacyl substitution upon PI3K binding as
compared to ZSTK474. It should be noted that in all cases, the
inhibitory potency is poorer against every isoform, but the loss
of affinity is more modest at PI3Kβ. The presence of the acidic
group D862 in PI3Kβ is effectively rescuing binding. The
conformational restriction engendered by the L-α-substituent
While improved cell permeability relative to ZSTK474 may
provide one explanation for this efficacy, other factors may be
contributing such as cellular metabolism or activity at other
PI3K pathway enzymes. In a screen (KinomeScan) of 21 versus
another 96 kinases, only the class II PI3K PI3KC2β showed
binding affinity comparable to that seen for PI3Kβ. The results
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Rominger, C. M.; Ariazi, J. L.; Sherk, C. S.; Schaber, M. D.; McSurdy-
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In summary, on the basis of the observed X-ray structure of
ZSTK474 in PI3Kδ, a series of L-aminoacylpiperazine-
substituted analogues have been prepared that exhibit excellent
potency and selectivity for PI3Kβ isoforms. The configuration
of the amino acids is pivotal to the selectivity, underpinning a
well-defined interaction with the nonconserved binding site
residue D862. The inhibitors show an alternate mechanistic
basis for selectivity in comparison to other recently reported
selective inhibitors. The compounds show inhibition of PI3Kβ-
dependent function in PTEN-deficient cancer cells and
effectively inhibit growth of the cell line. The compounds
provide a basis for the further study of PI3Kβ function in a
number of disease contexts.
ASSOCIATED CONTENT
* Supporting Information
■
S
Full experimental details relating to the synthesis of compounds
and methods for biochemical and cellular assays. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Corresponding Author Philip Thompson; Tel: +613
99039672; Fax +613 99039582; email: philip.thompson@
monash.edu.
■
Funding
J.-A.P. and M.S.M. are recipients of Australian Postgraduate
Award (APA) Scholarships. M.S.M. was the recipient of a top-
up scholarship from the CRC for Cancer Therapeutics. This
work was funded through the National Institutes of Health
Grants CA43460 and CA62924, the Virginia and D.K. Ludwig
Fund for Cancer Research (USA), the Cancer Council Victoria
No. 436708, and a National Health and Medical Research
Council Grant No. 545943 (Australia).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
ATP, adenosine triphosphate; Fmo, fluorenylmethyloxycarbon-
yl; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and
tensin homologue
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