Small molecule inhibition of phosphatidylinositol- 3,4,5-triphosphate (PIP3) binding to pleckstrin homology domains Benchun Miaoa,1, Igor Skidanb,1, Jinsheng Yangc, Alexey Lugovskoyd,2, Mikhail Reibarkhd, Kai Longe, Tres Brazella, Kulbhushan A. Durugkarf, Jenny Makia, C. V. Ramanaf, Brian Schaffhausena, Gerhard Wagnerd, Vladimir Torchilinb, Junying Yuane, and Alexei Degtereva,3 aDepartment of Biochemistry, Tufts University School of Medicine, Boston, MA 02111; bDepartment of Pharmaceutical Sciences, Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA 02115; cNeuroscience Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129; Departments of dBiological Chemistry and Molecular Pharmacology and eCell Biology, Harvard Medical School, Boston, MA 02115; and fNational Chemical Laboratory, Pune 411008, India Edited by Philip N. Tsichlis, Tufts Medical Center, Boston, MA, and accepted by the Editorial Board October 13, 2010 (received for review April 11, 2010) The PI3-kinase (PI3K) pathway regulates many cellular processes, downstream from growth factor and oncogene signals. A particu- especially cell metabolism, cell survival, and apoptosis. Phospha- larly important example of PIP3-dependent activation is that of tidylinositol-3,4,5-trisphosphate (PIP3), the product of PI3K activity serine-threonine kinase Akt. It is achieved both through the and a key signaling molecule, acts by recruiting pleckstrin-homology binding of Akt PH domain to PIP3 and membrane translocation of (PH) domain-containing proteins to cell membranes. Here, we de- another target of PIP3, PDK1, which phosphorylates and activates scribe a new structural class of nonphosphoinositide small molecule Akt. The Akt family plays a fundamental role in cell survival, antagonists (PITenins, PITs) of PIP3–PH domain interactions (IC50 growth, and energy metabolism (1, 7). ranges from 13.4 to 31 μM in PIP3/Akt PH domain binding assay). Although lipid–protein interactions mediate PI3K signaling and PITs inhibit interactions of a number of PIP3-binding PH domains, are frequently deregulated in cancer, most therapeutic strategies including those of Akt and PDK1, without affecting several PIP2- targeting the PI3K pathway have focused on inhibitors for down- selective PH domains. As a result, PITs suppress the PI3K-PDK1-Akt stream targets, including PDK1 (8) and Akt (9). Phospholipid– pathway and trigger metabolic stress and apoptosis. A PIT-1 analog protein interactions have not been as actively targeted, even displayed significant antitumor activity in vivo, including inhibition though lipid molecules are among the most important classes of of tumor growth and induction of apoptosis. Overall, our studies second messengers. It is surprising considering that they represent demonstrate the feasibility of developing specific small molecule “prototypic” small molecule–protein interactions usually involving antagonists of PIP3 signaling. well-defined binding sites (10). Conceptually, protein–lipid inter- actions may be more readily targetable compared with protein– PIP3 antagonist | anticancer protein interactions, which frequently involve interactions of ex- tended flat protein surfaces difficult to disrupt by small molecules. fi ysregulation of the phosphoinositide 3-kinase (PI3K) path- Here, we have pursued the identi cation of the small molecule Dway has been implicated in many human diseases. Hyper- antagonists of PIP3/protein binding. We report the discovery of activation of this pathway is known to play an important role in selective nonphosphoinositide PIP3 inhibitors, termed PITenins ≈ tumorigenesis, whereas deficiencies contribute to the development (PITs). PIT-1, selected in a screen of 50,000 small molecules by of type II diabetes. Therefore, this pathway offers promising tar- using a PIP3/Akt PH domain binding assay, has been extensively gets for the development of drugs to combat these diseases (1, 2). characterized. Our studies demonstrate that PIT-1 effectively fi Class I PI3Ks (α, β, and γ) are recruited to the plasma mem- inhibits cancer cell survival and induces cell apoptosis by speci - cally inhibiting PIP3-dependent PI3K-PDK1-Akt signaling, re- brane in response to growth factor and hormone stimulation fi to mediate the phosphorylation of lipid phosphatidylinositol- sulting in signi cant antitumor activity in vivo. Thus, we show not only a methodology usable to identify additional classes of 4,5-bisphosphate (PIP2), generating phosphatidylinositol-3,4,5- – trisphosphate (PIP3), which orchestrates multiple downstream phospholipid protein interactions, but also the feasibility of de- veloping cell-permeable specific small molecule nonlipid antago- intracellular signaling events (2). PIP3 signaling is terminated by – the phosphatase PTEN, which dephosphorylates PIP3. Genetic nists of lipid protein interactions. alterations targeting PTEN are among the most frequent muta- Results tions in human cancers, indicating a critical role of uncontrolled Identification of PIT-1 as a Specific PIP3 Antagonist. To identify signaling through PIP3 in tumorigenesis and metastasis (3). This compounds that disrupt the interaction between PIP3 and PH conclusion is reinforced by transgenic studies establishing that domains, we developed a high-throughput fluorescence polari- loss of PTEN leads to tumorigenesis (1). PIP3 controls a complex cellular signaling network regulating cell growth, proliferation, and survival. PIP3 target proteins are Author contributions: B.M., C.V.R., B.S., G.W., V.T., J.Y., and A.D. designed research; B.M., located in the cytosol of unstimulated cells and are recruited to the I.S., J.Y., A.L., M.R., K.L., T.B., K.A.D., and J.M. performed research; B.M., I.S., J.Y., A.L., membrane through pleckstrin-homology (PH) domain-mediated M.R., K.L., K.A.D., C.V.R., and A.D. analyzed data; and B.M. and A.D. wrote the paper. binding to newly formed PIP3. Membrane translocation and acti- The authors declare no conflict of interest. vation of the PIP3 target proteins initiate a variety of local re- This article is a PNAS Direct Submission. P.N.T. is a guest editor invited by the Editorial sponses, including assembly of signaling complexes and priming Board. of protein kinase cascades (1, 2). PIP3 regulates an array of PH 1B.M. and I.S. contributed equally to this work. domain-containing proteins (4), such as serine-threonine kinases 2Present address: Drug Discovery Department, Biogen IDEC Inc., 14 Cambridge Center, Akt and PDK1, GRP1, a GDP/GTP exchange factor of ADP Cambridge, MA 02142. ribosylating factor 6, and protein tyrosine kinases of the Bruton’s 3To whom correspondence should be addressed. E-mail: [email protected]. tyrosine kinase (Btk) and Tec families (5, 6). This diversity in PIP3 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. signaling makes it one of the most important second messengers 1073/pnas.1004522107/-/DCSupplemental. 20126–20131 | PNAS | November 16, 2010 | vol. 107 | no. 46 www.pnas.org/cgi/doi/10.1073/pnas.1004522107 Downloaded by guest on September 29, 2021 zation (FP)-binding assay by using recombinant 1–123 amino acid binding of PI-3,4-P2 to Akt PH domain (Fig. S1H), consistent with N-terminal fragment of human Akt1, encompassing the PH do- the notion that PITs targeting Akt PH domain binding pocket, main (PH123 protein), and a fluorescent NBD-labeled PIP3 rather than a lipid. These data suggest that PIT-1 and PIT-2 rep- molecule. The R86A mutation in the PH domain, shown to disrupt resent unique nonphosphoinositide-related selective antagonists of PIP3–PH interaction (11), completely abrogated fluorescent PIP3 PIP3/PH domain binding. binding by PH123 (Fig. S1A), validating the assay. Our screen of To characterize the mode of action of PIT-1, we performed ≈50,000 diverse small molecules using this FP assay identified two NMR-based analysis of PIT-1 binding to the PH domain of Akt. We distinct inhibitors of PIP3/PH domain binding, which were termed found PIT-1 induces significant changes in the 2D HSQC 1H-15N PIT-1 and PIT-2 (Fig. 1A). These molecules disrupted PIP3/Akt correlation spectrum of Akt PH123, indicative of the direct PIT-1/ A PH domain binding with IC50 = 31.03 and 31.52 μM for PIT-1 and Akt PH domain binding (Fig. S2 ). We titrated Akt PH123 with PIT-2, respectively (Fig. 1B). Although giving similar results to PIT-1 to identify the residues affected by binding. Using published PIT-1, PIT-2 displayed limited stability and was used primarily to Akt2 HSQC peak assignments (13), we observed that the residues confirm the major conclusions. Analysis of commercial PIT-1 W22, Y26, and N54, located in the PIP3 binding pocket of the Akt analogs suggested that thiourea and hydroxyl groups are critical for PH domain, are affected by PIT-1. We found that PIT-1 had no the activity in vitro and revealed closely related inactive analogs of effect on the R86A PH domain mutant, which lacks PIP3 binding PIT-1, termed PIT-1i-1 and PIT-1i-2 (Fig. 1A), which have been activity, confirming the PIP3-mimetic nature of PIT-1. Using this used as negative controls. Besides the Akt PH domain, PIT-1 and information, we propose a binding model of PIT-1 on the surface of PIT-2 also inhibited the binding of PIP3 to the PH domains of the Akt PH domain in which the PIT-1 binding site overlaps with PDK1, GRP1, and ARNO with lower affinity (Fig. 1B and Fig. S1 the PIP3 binding site. Consistent with NMR data, W22 and Y26 B, C,andF) and failed to inhibit PIP3 binding to the PIP3-specific flank the RPR motif that interacts with the 3-phosphate of PIP3 PH domain of Btk (Fig. 1B and Fig. S1G). Thus, PITs exhibited and the nitro group of PIT-1, whereas N54 packs against the ter- selectivity toward a distinct subset of PIP3-specific PH domains. minal phenyl group of PIT-1 and does not interact with PIP3 (Fig. Furthermore, PITs failed to inhibit PIP2-specific interactions be- S2 B and C).