Crystal structures of PI3Kα complexed with PI103 and its derivatives: New directions for inhibitors design

Article abstract of DOI:10.1021/ml400378e

The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays important roles in cell proliferation, growth, and survival. Hyperactivated PI3K is frequently found in a wide variety of human cancers, validating it as a promising target for cancer therapy. We determined the crystal structure of the human PI3Kα-PI103 complex to unravel molecular interactions. Based on the structure, substitution at the R₁ position of the phenol portion of PI103 was demonstrated to improve binding affinity via forming a new H-bond with Lys802 at the bottom of the ATP catalytic site. Interestingly, the crystal structure of the PI3Kα-9d complex revealed that the flexibility of Lys802 can also induce additional space at the catalytic site for further modification. Thus, these crystal structures provide a molecular basis for the strong and specific interactions and demonstrate the important role of Lys802 in the design of novel PI3Kα inhibitors.

Full text of DOI:10.1021/ml400378e

Letter
pubs.acs.org/acsmedchemlett
Crystal Structures of PI3Kα Complexed with PI103 and Its
Derivatives: New Directions for Inhibitors Design
Yanlong Zhao,†,‡ Xi Zhang,§,‡ Yingyi Chen,†,‡ Shaoyong Lu,†,‡ Yuefeng Peng,∥,‡ Xiang Wang,§
Chengliang Guo,§ Aiwu Zhou,† Jingmiao Zhang,† Yu Luo,† QianCheng Shen,† Jian Ding,§
Linghua Meng,§,* and Jian Zhang†,*
†Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai
JiaoTong University, School of Medicine, Shanghai 200025, China
§Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, China
∥Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology (SIAT), Chinese Academy of Sciences
(CAS), Guangdong, Shenzhen 518055, China
S
* Supporting Information
ABSTRACT: The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays important
roles in cell proliferation, growth, and survival. Hyperactivated PI3K is frequently found in a
wide variety of human cancers, validating it as a promising target for cancer therapy. We
determined the crystal structure of the human PI3Kα−PI103 complex to unravel molecular
interactions. Based on the structure, substitution at the R1 position of the phenol portion of
PI103 was demonstrated to improve binding affinity via forming a new H-bond with Lys802 at
the bottom of the ATP catalytic site. Interestingly, the crystal structure of the PI3Kα−9d
complex revealed that the flexibility of Lys802 can also induce additional space at the catalytic
site for further modification. Thus, these crystal structures provide a molecular basis for the strong and specific interactions and
demonstrate the important role of Lys802 in the design of novel PI3Kα inhibitors.
KEYWORDS: PI3K, PI103, crystal structure, drug design, cancer therapy
With multiple ongoing efforts in academic and industrial
organizations to develop clinically relevant inhibitors against
PI3K, a number of inhibitors have already entered clinical
trials.2,10 PI103 is one of the first synthesized PI3K inhibitors; it
belongs to the pyridinylfuranopyrimidine class and inhibits PI3K
in an ATP-competitive manner with selectivity toward PI3Kα.11
PI103 has already demonstrated significant antitumor activity
against several human tumor xenografts, especially those with
well-established abnormalities in the PI3K pathway.12 Although
PI103 failed to enter clinical trials, GDC0941, a drug candidate
derived from PI103, is in phase I/II trials.7 Thus, PI103 provides
a promising chemical template for the discovery and develop-
ment of PI3K inhibitors. However, the precise interactions
between PI103 and PI3Kα remain unknown. A better under-
standing of the interactions between PI103 and PI3K will
facilitate the rational design of PI3K inhibitors with improved
potency and selectivity.
he lipid kinase family of phosphatidylinositol 3-kinases
T(PI3Ks) plays pivotal roles in many cellular processes,
including proliferation, survival, differentiation, and metabo-
lism.1−3 Class I PI3K, the best physiologically, biochemically, and
structurally characterized member of the PI3K family, consists of
four isoforms, α, β, γ, and δ. Each isoform is a heterodimer that
comprises a p110 catalytic subunit and a p85 regulatory subunit.
Upon insulin and growth factor stimulation, PI3Ks phosphor-
ylate phosphatidylinositol-3,4-bisphosphate (PIP2) to produce
phosphatidylinositol-3,4,5-triphosphate (PIP3). The cellular
level of PIP3 is also tightly regulated by phosphatases, such as
the phosphatase and tensin homologue (PTEN), which
dephosphorylates PIP3 back to PIP2.4,5 The PI3K pathway is
frequently deregulated in a wide range of tumor types as a result
of hyperactivation of upstream growth factor signaling, mutation,
or loss of PTEN,6 and oncogenic mutations in PIK3CA,7 which
provides further evidence of the role of PI3K in tumorigenesis.
Moreover, accumulating evidence indicates that hyperactivation
of PI3Kα is inextricably linked to cancer survival and resistance to
existing therapies in a great proportion of human cancers.8
Therefore, targeting PI3Ks with small-molecular-weight inhib-
itors provides an attractive opportunity for cancer therapy and
for overcoming resistance to current therapies, and thus,
significant efforts have recently been made to develop PI3K
inhibitors.9
To date, the solved PI3Kα−inhibitors crystal structures are
extremely scarce and the nSH2 and iSH2 domains are not
complete in only three of these structures (Supporting
Information Table S1).13−15 To generate human wild-type full-
length p110α and the nSH2 and iSH2 domains (hereafter termed
Received: September 23, 2013
Accepted: December 10, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
“niSH2”) of p85α that would be suitable for crystallography, a
fusion protein of the 6*his-TEV-p85α (318−615)-linker-p110α
was expressed in the Bac-to-Bac Baculovirus expression system
(Invitrogen) using a construct containing the iSH2 domain
(467−568) of p85α fused at the N-terminus of the full-length
p110α, as described previously.15,16 To produce a stoichiometric
complex to enhance crystallization, analogous niSH2−p110α
fusion constructs were designed, incorporating an additional
linker between the iSH2 domain and the p110α in place of the
thrombin cleavage site, which is prone to nonspecific proteolytic
cleavage and accompanying heterogeneity. Using this method, a
high purity of the 6*his-TEV-p85α (318−615)-linker-p110α
homogeneous fusion protein was obtained via Ni-NTA,
Sepharose Q, and gel filtration. The resulting complex contained
all five p110α domains [an amino-terminal adaptor-binding
domain (ABD), residues 1 to 108; a Ras-binding domain (RBD),
residues 190 to 291; a C2 (protein-kinase-C homology-2)
domain, residues 330 to 480; a helical domain, residues 525 to
696; and a carboxyl-terminal kinase domain, residues 697 to
1068], as well as the nSH2 (residues 318 to 430) and iSH2
(residues 431 to 615) domains of p85α (Figure 1). Through a
residues Ile800, Asp810, Tyr836, Glu849, and Val851 on one
side and residues Met922, Ile932, and Asp933 on the other side
(Figure 2C). Three H-bonds are formed between PI103 and the
active site residues of p110α. The morpholine oxygen donates an
H-bond to the amide of the hinge Val851. The hydroxyl group of
the phenol moiety makes two H-bonds to the carboxyl group of
Asp810 and to the hydroxyl group of Tyr836.
Upon closer inspection of the crystal structure of the p110α/
p85α−PI103 complex, we noted that the distance between the
phenol moiety of PI103 and the positively charged residue
Lys802 is 3.96 Å, which may accommodate small substituted
groups toward the side chain of Lys802 at the bottom of the ATP
catalytic site for further modification to generate new PI103
derivatives with desirable potencies (Figure 2D). Based on this
hypothesis, a series of substituted groups on the meta-site (R1
group in Table 1) of the phenol moiety in PI103 were introduced
and probed the potential space (Supporting Information Scheme
S1). As shown in Table 1, PI103 derivatives 9a, 9b, and 9c, in
which a fluoro, chloro, and bromo group with different radius was
replaced, respectively, for the original hydrogen atom in the R1
position of PI103, failed to enhance potency through the
additional interactions with Lys802 (Figure 3A); based on the
molecular modeling, we speculated that compound 9d, a slightly
larger amino group with a positive charge in the R1 position,
would cause a decreased potency due to the electrostatic
repulsion between the incoming NH2 substitute and Lys802.
Unexpectedly, compound 9d was as potent as PI103 against the
PI3Kα (Figure 3B); substitution of the NH2 group in compound
9d with an OH group led to compound 9e, in which the oxygen
atom acceptor was capable of forming an H-bond with Lys802.
Moreover, molecular modeling using the crystal structure of the
p110α/p85α−PI103 complex ascertained that the incoming OH
group was hydrogen-bonded to the side chain of Lys802 (Figure
3C). Indeed, compound 9e was approximately 3-fold more
potent against PI3Kα compared with PI103, with an IC50 value of
5.9 nM. Finally, large substituted groups on the R1 position of
PI103 were further explored for the space. Replacing the
hydrogen atom in the R1 position of PI103 with a nitro
(compound 9f), methyl (compound 9g), and trifluoromethyl
(compound 9h) group revealed that these compounds were
negligible to inhibit the kinase activity of PI3Kα (Table 1). These
results suggest that introducing a bulky group in the R1 position
may produce dramatic steric clashes at the bottom of the ATP
catalytic site.
Figure 1. Overview of the p110α/niSH2 heterodimer. (A) Scheme of
the domain organization. The same color coding is used throughout this
figure. Gray regions are linkers between domains. Residue range for each
domain is labeled under the domain diagram. (B) Diagram of the
p110α/niSH2 heterodimer. The PI103 bound in the kinase domain is
shown as spheres. (C) Surface diagram of the p110α/niSH2
heterodimer, alternate view. The PI103 bound in the kinase domain is
shown as sticks.
Structurally, Met772 exists in the flexible loop of PI3Kα, which
is in an “up” conformation in the apo PI3Kα and a “down”
conformation when PI103 is bound to the active site of PI3Kα. In
the PI3Kα−PI103 complex, the hydroxyl group of PI103 forms
bifurcated H-bonds with Asp810 and Tyr836 (Figure 2C), and
Met772 interacts with PI103 via hydrophobic interactions
(Supporting Information Figure S1). To investigate the effect
of these residues on the activity of PI103 and its derivatives,
M772A, D810A, and Y836A mutants of PI3Kα were generated,
and the activity of PI103 and compound 9e against these mutants
was determined. As shown in Table 2, compound 9e was more
potent than PI103 against all three mutants, in particular the
M772A and Y836A mutants; the fold change, defined by the ratio
of the IC50 values of PI103 and compound 9e, was 3.0 for the wild
type kinase and increased to 19.4 and 5.7 in the M772A and
Y836A mutants, respectively (Table 2). These differences are
most likely attributed to the extra hydrogen bonding interaction
between the hydroxyl group in the R1 position of compound 9e
sparse matrix screening of 1400 conditions and seeding
optimization, a diffraction-quality crystal was produced for data
collection, and an apo crystal was also incubated with 10 mM
PI103. Diffraction data to a resolution of 2.6 Å were obtained for
both the apo crystals and the PI103-containing ones and were
refined to Rwork/Rfree values of 21.7/27.4 and 21.5/27.3,
respectively (Supporting Information Table S2). Both structures
maintained an overall triangle shape, with the long coiled-coil of
iSH2 forming the base (Figure 1B and C).
The crystal structure of apo p110α revealed that the ATP-
binding pocket is located in a cleft between the N- and C-
terminal lobes of the kinase catalytic domain of p110α (Figure
2A), similar to the ATP binding sites in other protein kinases,
with many of the enzyme/ATP interactions involving residues in
the linker region between the two lobes. The crystal structure of
p110α/p85α−PI103 appeared to verify that PI103 binds to the
ATP-binding site on the p110α kinase domain (Figure 2B).
PI103 adopts a flat conformation and sits between p110α
B
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ACS Medicinal Chemistry Letters
Letter
Figure 2. Binding pocket of p110α complexed with PI103. (A) Binding pocket of X-ray apo p110α (PDB ID: 4L1B); (B) Electron density map (2Fobs
−
Fcalc) of PI103 rendered at 1.0σ is shown in the binding pocket of the kinase domain; (C) X-ray complex of PI103 to p110α (PDB ID: 4L23). The
structure of PI103 is shown as a stick representation, and the key binding site residues are shown as sticks. Hydrogen bonds between PI103 and the
protein are shown as a dashed line; (D) Surface representation of PI103 bound p110α. Arrow indicates the direction of PI103 modification.
and Lys802, which may stabilize the binding between compound
9e and the mutated PI3Kα.
Table 1. Effects of Test Compounds on the Kinase Activity of
Class I PI3Kα
a
To determine the binding paradigms of PI103 derivatives to
the p110α kinase catalytic domain, we incubated compounds 9a,
9d, and 9e with the p110α/p85α complex using a similar
protocol for crystallization of the p110α/p85α−PI103 complex.
After screening and optimization, a 2.8 Å resolution crystal
structure of the human p110α/p85α in complex with compound
9d was obtained (Supporting Information Table S2). As shown
in Figure 3B, generally, the interaction of compound 9d with the
ATP catalytic site of p110α/p85α was similar to its interaction
with the p110α/p85α−PI103 complex. However, the locally
pronounced difference between the two crystal complexes was
associated with the conformation of Lys802. The Lys802 side
chain rotated away from the ATP catalytic site in the 110α/
p85α−9d complex, whereas it pointed to the phenol moiety of
PI103 in the ATP catalytic site in the 110α/p85α−PI103
complex (Supporting Information Figure S2). The induced
conformation of the Lys802 side chain in the 110α/p85α−9d
complex led to the vast space approaching the amino-substituted
phenol moiety of 9d (Figure 3D), which can accommodate
various large substituted groups in the cavity and provide a
potential new direction to design more potent and selectivity
inhibitors against PI3Kα based on compound 9d.
b
cmpd
R1
PI3KαIC50 (nM)
PI103
9a
H
F
17.9 1.9
40.9 5.8
990.0 119.1
1210.0 246.8
20.6 10.2
5.9 0.4
9b
9c
Cl
Br
9d
9e
−NH2
−OH
−NO2
−CH3
−CF3
9f
>10,000
9g
>10,000
9h
>10,000
a
Effects of test compounds on the kinase activity of class I PI3K were
b
In summary, our PI3Kα−PI103 crystal complex demonstrated
that the introduction of apt substitutes at the R1 position of the
phenol moiety of PI103 results in derivatives with enhanced
potencies, confirmed by compound 9e as a better analogue by
assessed as described in the Experimental Section. IC50 values shown
are the average of least three independent experiments in duplicate
with typical variations of less than 20%.
C
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ACS Medicinal Chemistry Letters
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Figure 3. Binding modes of (A) compound 9a from docking; (B) compound 9d from X-ray (PDB ID: 4L2Y); (C) compound 9e from docking; (D)
potential cavity of p110α induced by the binding of 9d. Key residues surrounding the active site of p110α are labeled, and carbon atoms are colored in
pale green.
Accession Codes
Table 2. Effects of Compound 9e and PI103 on the Kinase
Activities of Wild Type and Mutated PI3Kα
a
Coordinates for the apo PI3Kα, PI3Kα−PI103, and PI3Kα−9d
structures have been deposited in the Protein Data Bank with
access codes 4L1B, 4L23, and 4L2Y.
b
cmpd (IC50, nM)
c
PI3Kα
PI103
9e
5.9
fold change
wt
17.9
1034.4
74.0
3.0
19.4
1.2
AUTHOR INFORMATION
M772A
D810A
Y836A
53.4
61.3
■
Corresponding Authors
796.3
139.8
5.7
*E-mail: jian.zhang@sjtu.edu.cn.
*E-mail: lhmeng@simm.ac.cn.
a
Mutated PI3Kα were expressed and purified as described in the
b
Experimental Section. IC50 values shown are the average of at least
three independent experiments performed in duplicate with typical
variations of less than 20%. The fold change was obtained from the
Author Contributions
c
‡The manuscript was written through contributions of all
authors. Y.Z., X.Z., Y.C., S.L., and Y.P. contributed equally.
ratio of the IC50 value of PI103 to that of compound 9e.
Funding
forming a new H-bond with Lys802. Moreover, 9e was more
tolerant to mutations of PI3Kα in the ATP catalytic site, such as
M772A, D810A, and Y836A. Specially, the additional space near
the phenol moiety was induced by a conformational change of
Lys802 in the ATP catalytic site of the PI3Kα−9d crystal
complex, which can be utilized for further design and
optimization.
This work was supported by the Program for New Century
Excellent Talents in University (NCET-12-0355), the Shanghai
Rising-Star Program (13QA1402300), the National Science &
Technology Major Project “Key New Drug Creation and
Manufacturing Program” (2012ZX09301-001), the National
Natural Science Foundation of China (81322046, 81302698,
81021062, 21002062, and 21102090), and the Knowledge
Innovation Program of Chinese Academy of Sciences (KSCX2-
EW-Q-3).
ASSOCIATED CONTENT
■
S
* Supporting Information
Expression, purification, and crystallization of PI3Kα, synthetic
procedures, assay details, and molecular modeling. This material
is available free of charge via the Internet at http://pubs.acs.org.
Notes
The authors declare no competing financial interest.
D
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ACS Medicinal Chemistry Letters
Letter
potent and isoform-selective targeted covalent inhibitor of the lipid
kinase PI3Kα. J. Med. Chem. 2013, 56, 712−721.
(16) Huang, C. H.; Mandelker, D.; Schmidt-Kittler, O.; Samuels, Y.;
Velculescu, V. E.; Kinzler, K. W.; Vogelstein, B.; Gabelli, S. B.; Amzel, L.
M. The structure of a human p110α/p85α complex elucidates the effects
of oncogenic PI3Kα mutations. Science 2007, 318, 1744−1748.
ACKNOWLEDGMENTS
■
We thank Prof. Shaomeng Wang at the University of Michigan
for fruitful discussions on the design of PI103 derivatives.
ABBREVIATIONS
■
PI3K, phosphatidylinositol 3-kinase; ATP, adenosine triphos-
phate; PIP2, phosphatidylinositol-3,4-bisphosphate; PIP3, phos-
phatidylinositol-3,4,5-triphosphate; PTEN, phosphatase and
tensin homologue; ABD, amino-terminal adaptor-binding
domain; RBD, Ras-binding domain
REFERENCES
■
(1) Camps, M.; Ruckle, T.; Ji, H.; Ardissone, V.; Rintelen, F.; Shaw, J.;
̈
Ferrandi, C.; Chabert, C.; Gillieron, C.; Franco
̧
n, B.; Martin, T.;
Gretener, D.; Perrin, D.; Leroy, D.; Vitte, P. A.; Hirsch, E.; Wymann, M.
P.; Cirillo, R.; Schwarz, M. K.; Rommel, C. Blockade of PI3Kγ
suppresses joint inflammation and damage in mouse models of
rheumatoid arthritis. Nat. Med. 2005, 11, 936−943.
(2) Knight, Z. A.; Gonzalez, B.; Feldman, M. E.; Zunder, E. R.;
Goldenberg, D. D.; Williams, O.; Loewith, R.; Stokoe, D.; Balla, A.;
Toth, B.; Balla, T.; Weiss, W. A.; Williams, R. L.; Shokat, K. M. A
pharmacological map of the PI3-K family defines a role for p110α in
insulin signaling. Cell 2006, 125, 733−747.
(3) Fan, Q. W.; Knight, Z. A.; Goldenberg, D. D.; Yu, W.; Mostov, K.
E.; Stokoe, D.; Shokat, K. M.; Weiss, W. A. A dual PI3 kinase/mTOR
inhibitor reveals emergent efficacy in glioma. Cancer Cell 2006, 9, 341−
349.
(4) Castellino, R. C.; Muh, C. R.; Durden, D. L. PI-3 kinase-PTEN
signaling node: an intercept point for the control of angiogenesis. Curr.
Pharm. Des. 2009, 15, 380−388.
(5) Foster, J. G.; Blunt, M. D.; Carter, E.; Ward, S. G. Inhibition of
PI3K signaling spurs new therapeutic opportunities in inflammatory/
autoimmune diseases and hematological malignancies. Pharmacol. Rev.
2012, 64, 1027−1054.
(6) Davies, M. A. The role of the PI3K-AKT pathway in melanoma.
Cancer J. 2012, 18, 142−147.
(7) Willems, L.; Tamburini, J.; Chapuis, N.; Lacombe, C.; Mayeux, P.;
Bouscary, D. PI3K and mTOR signaling pathways in cancer: new data
on targeted therapies. Curr. Oncol. Rep. 2012, 14, 129−138.
(8) Sabbah, D. A.; Brattain, M. G.; Zhong, H. Dual inhibitors of PI3K/
mTOR or mTOR-selective inhibitors: which way shall we go? Curr. Med.
Chem. 2011, 18, 5528−5534.
(9) Chen, Y.; Wang, B. C.; Xiao, Y. PI3K: a potential therapeutic target
for cancer. J. Cell Physiol. 2011, 227, 2818−2821.
(10) Denny, W. A. Phosphoinositide 3-kinase α inhibitors: a patent
review. Expert Opin. Ther. Pat. 2013, 23, 789−799.
(11) Knight, Z. A.; Chiang, G. G.; Alaimo, P. J.; Kenski, D. M.; Ho, C.
B.; Coan, K.; Abraham, R. T.; Shokat, K. M. Isoform-specific
phosphoinositide-3 kinase inhibitors from an arylmorpholine scaffold.
Bioorg. Med. Chem. 2004, 12, 4749−4759.
(12) Fan, Q.-W.; Cheng, C.-K.; Nicolaides, T. P.; Hackett, C. S.;
Knight, Z. A.; Shokat, K. M.; Weiss, W. A. A dual phosphoinositide-3-
kinase alpha/mTOR inhibitor cooperates with blockade of epidermal
growth factor receptor in PTEN-mutant glioma. Cancer Res. 2007, 67,
7960−7965.
(13) Mandelker, D.; Gabelli, S. B.; Schmidt-Kittler, O.; Zhu, J.;
Cheong, I.; Huang, C. H.; Kinzler, K. W.; Vogelstein, B.; Amzel, L. M. A
frequent kinase domain mutation that changes the interaction between
PI3K and the membrane. Proc. Natl. Acad. Sci. U. S. A. 2009, 106,
16996−7001.
(14) Hon, W. C.; Berndt, A.; Williams, R. L. Regulation of lipid binding
underlies the activation mechanism of class IA PI3-kinases. Oncogene
2012, 31, 3655−3666.
(15) Nacht, M.; Qiao, L.; Sheets, M. P., St; Martin, T.; Labenski, M.;
Mazdiyasni, M.; Karp, R.; Zhu, Z.; Chaturvedi, P.; Bhavsar, D.; Niu, D.;
Westlin, W.; Petter, R. C.; Medikonda, A. P.; Singh, J. Discovery of a
E
dx.doi.org/10.1021/ml400378e | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX