PI-273, a Substrate-Competitive, Specific Small- Molecule Inhibitor of Pi4kiia, Inhibits the Growth of Breast Cancer Cells

Published OnlineFirst August 21, 2017; DOI: 10.1158/0008-5472.CAN-17-0484

Cancer Therapeutics, Targets, and Chemical Biology Research

PI-273, a Substrate-Competitive, Speciﬁc Small- Molecule Inhibitor of PI4KIIa, Inhibits the Growth of Breast Cancer Cells Jiangmei Li1,2,ZhenGao1,3, Dan Zhao3,4, Lunfeng Zhang1,3, Xinhua Qiao1,3, Yingying Zhao1,2, Hong Ding4, Panpan Zhang2,3,5, Junyan Lu4, Jia Liu2, Hualiang Jiang4, Cheng Luo4,6, and Chang Chen1,3,7

Abstract

While phosphatidylinositol 4-kinase (PI4KIIa)hasbeen substrate phosphatidylinositol (PI), which contrasted with identified as a potential target for antitumor therapy, the clinical most other PI kinase inhibitors that bind the ATP binding site. applications of PI4KIIa are limited by a lack of specificinhibi- PI-273 reduced PI4P content, cell viability, and AKT signaling in tors.Herewereportthefirst small-molecule inhibitor wild-type MCF-7 cells, but not in PI4KIIa knockout MCF-7 cells, (SMI) of human PI4KIIa. Docking-based and ligand-based indicating that PI-273 is highly selective for PI4KIIa.Mutant virtual screening strategies were firstemployedtoidentify analysis revealed a role of palmitoylation insertion in the promising hits, followed by two rounds of kinase activity selectivity of PI-273 for PI4KIIa. In addition, PI-273 treatment inhibition validation. 2-(3-(4-Chlorobenzoyl)thioureido)-4- retarded cell proliferation by blocking cells in G2–M, inducing ethyl-5-methylthiophene-3-carboxamide (PI-273) exhibited cell apoptosis and suppressing colony-forming ability. Impor- the greatest inhibitory effect on PI4KIIa kinase activity tantly, PI-273 significantly inhibited MCF-7 cell-induced breast (IC50 ¼ 0.47 mmol/L) and suppressed cell proliferation. Surface tumor growth without toxicity. PI-273 is the first substrate- plasmon resonance and thermal shift assays indicated that PI- competitive, subtype-specific inhibitor of PI4KIIa,theuseof 273 interacted directly with PI4KIIa. Kinetic analysis identified which will facilitate evaluations of PI4KIIa as a cancer thera- PI-273 as a reversible competitive inhibitor with respect to the peutic target. Cancer Res; 77(22); 6253–66. 2017 AACR.

Introduction diseases, including cancer (2–4), malaria (5), Alzheimer disease (6–8), and diabetes (9). Among the phosphatidylinositol Phosphatidylinositol (PI) is an essential phospholipid that kinases, PI4KIIa is targeted to the trans-Golgi network (TGN) serves as a metabolic precursor for both phosphoinositides and and endosomes. In mammalian cells, PI4KIIa is the dominant inositol (Ins) phosphates (1), and its related phosphatidylino- lipid kinase generating PI4-phosphate (PI4P; ref. 10), which is sitol kinases (PIK) play important roles in a number of human not only the precursor of the important regulatory phosphoinositides PI(4,5)P2 and PI(3,4,5)P3 but also is an emerging

1 regulatory molecule in trans-Golgi (11) and endosomal traf- National Laboratory of Biomacromolecules, CAS Center for Excellence in fi Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, cking (12, 13), as well as phagocytosis (14). PI4KIIa knockout Chaoyang District, Beijing, China. 2Shanghai Institute for Advanced Immuno- mice develop late-onset neurologic features that resemble chemical Studies, ShanghaiTech University, Shanghai, China. 3University of human hereditary spastic paraplegia (15). Recently, an increas- Chinese Academy of Sciences, Shijingshan District, Beijing, China. 4Drug Dis- ing number of reports have identified PI4KIIa as a potential covery and Design Center, State Key Laboratory of Drug Research, Shanghai target for breast cancer therapy (3, 4, 16–19). We previously Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China. reported that increased PI4KIIa expression promotes tumor 5Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China. 6The State Key Laboratory of Toxicology and Medical Counter- growth by altering the HER2/PI3-kinase (PI3K)/ERK signaling measures, Academy of Military Medical Science, Beijing, China. 7Beijing Institute cascades (4) and that dual inhibition of EGFR and PI4KIIa for Brain Disorders, You An Men, Beijing, China. represents a novel strategy to combat EGFR-dependent tumors Note: Supplementary data for this article are available at Cancer Research (17). PI4KIIa is also important for the Wnt (20, 21) and Akt (3) Online (http://cancerres.aacrjournals.org/). signaling pathways. However, in contrast to the extensive study fi J. Li, Z. Gao, and D. Zhao contributed equally to this article. on PI3Ks, pharmacologic manipulation and basic scienti c research on PI4KIIa has been limited, largely owing to a lack Corresponding Authors: Chang Chen, National Laboratory of Biomacromole- of specific inhibitors. cules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, P.R. China. Phone: 8610-6488-8406; Fax: Numerous small-molecule inhibitors (SMI) of PIKs have 8610-6487-1293; E-mail: [email protected]; and Cheng Luo, Drug been identified via screening in the past two decades, but most Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai are ATP-competitive inhibitors of PI3Ks (22). Only 2-methyl-5- Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi nitro-2-[(6-bromoimidazo[1,2-a]pyridin-3-yl)methylene]-1- Road, Pudong District, Shanghai, 201203, PR China. Phone: 86-21-5027-1399; methylhydrazide-benzenesulfonic acid (PIK-75; refs. 23, 24) E-mail: [email protected] and NCGC00012848-02 (NIH-12848; ref. 25) have been doi: 10.1158/0008-5472.CAN-17-0484 identified as substrate-competitive inhibitors. Isoform selec- 2017 American Association for Cancer Research. tivity is complicated by high conservation of the nucleotide-

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binding pocket amino acid sequences of the different (30–32) and Autodock 4.2 (33, 34) programs were evaluated. PIKisoforms,thesiteatwhichmostSMIsbind.Because The substituent PI molecule was mixed with 50 decoy mole- isoform-selective inhibitors may reduce toxicity by decreas- cules and docked into the PI-binding site using the three ing off-target effects, substrate-competitive inhibitors are software programs. Because the GLIDE 5.5 software provided urgently needed. the best PI substituent ranking, this program was used for By combining crystal structure data, molecular dynamics (MD) subsequent structural-based virtual screening. Docking studies simulations, and biochemical and mutagenesis studies, we recent- in Gold 5.0 were performed using the default genetic algorithm ly identified the putative PI-binding pocket for PI4KIIa (26). The (GA) parameters and automatic settings with 100% search PI-binding pocket is lined by residues E157, Y159, R181, L184, efficiency. The kinase scoring function implemented in Gold T307, D308, and L349 on the palmitoylation insertion and was applied in the docking calculations, and GA docking was activation loop. In addition to contributing to the PI-binding settobeterminatedifthebestthree solutions were all within pocket, the palmitoylation insertion also interacts with the mem- 1.5 Å root mean square deviations (RMSD) of each other. In brane via an amphipathic helix, a4, and any perturbation that Autodock 4.2, the 50 decoy molecules and the PI substituent weakens its membrane interaction significantly impairs its kinase were docked to PI4KIIa using the Lamarckian GA, and a 0.375 activity. This insertion does not occur in other PIKs, making it an Å grid spacing was set for the energetic map calculations. The ideal site for screening specific inhibitors. In addition, a high- number of GA runs was set to 10, and the standard precision throughput and nonisotopic assay for PI4-kinase activity has been (SP) GLIDE 5.5 mode was used. The protein structure was developed (27), thereby enabling screens for PI4KIIa-specific prepared using the Protein Preparation Wizard Workflow with inhibitors among large compound libraries. Structure-based vir- default settings. The PAL loop and activation loop were defined tual screening (SBVS) has become a powerful tool in the tool kit of asthePI-bindingsiteinwhichthedockinggridswerecreated medicinal chemists for rapidly enriching hits from large pools of (26). The 822 compounds were docked into the defined bind- virtual compound libraries (28). In this study, we identified a PI ingsiteandrankedonthebasisoftheirGLIDEscore(G-score). substrate-competitive SMI for PI4KIIa that notably inhibits tumor Considering both the docking scores and binding modes, 522 growth in a breast cancer model without toxicity. compounds were selected for further evaluation, which were then structurally clustered into 60 clusters based on their 2D molecular fingerprints using the Cluster Molecules module in Materials and Methods Pipeline Pilot 7.5. Two or three candidate molecules with Virtual screening relatively simple chemical structures and higher G-scores with- The SPECS database (http://www.specs.net) is a compound in each structural cluster were retained. Ultimately, a total of library of 199,970 single-synthesized, well-characterized small 142 compounds were selected and purchased for the first-round molecules. This database has been widely used in the study of biochemical assay. On the basis of the structures of the 7 active compound screening processes and drug discovery and was compounds identified by the first-round biochemical assay, the used as the ligand database in this study. To refine the database SHAFTS method was again utilized for scaffold hopping (29). A to include compounds with good drug-like properties [(molec- total of 53 candidates were purchased for the second-round ular weight 500, log P 5, number of hydrogen bond donors biochemical assay. 7, number of hydrogen bond acceptors 14), molecular solubility –7 (expressed as log S, where S is the solubility in Expression and purification for PI4KIIa, PI4KIIb, and PI4KIIIb mol/L), ADMET solubility –5 (base 10 logarithm of molar The constructs PI4KIIa,PI4KIIb, and PI4KIIIb were expressed solubility as predicted by regression), ADMET solubility level as GST fusion proteins in Escherichia coli BL21-CodonPlus 2 (assigns the molecule to one of seven solubility classes based (DE3) competent cells. The cells were grown at 37 Cuntilan on the ADMET solubility value)], we filtered the database using OD600 of 1.0 was reached, at which point they were induced by Pipeline Pilot 7.5 (Scitegic, Inc.) prior to screening. The filtered 0.3 mmol/L isopropyl b-D-thiogalactoside at 16 Cfor18hours. database was used for a subsequent SHAFTS-3D ligand simi- The cells were then harvested and resuspended in buffer con- larity search. The SHAFTS algorithm was implemented in the taining 50 mmol/L HEPES (pH 7.5), 1 mol/L NaCl, 2 mmol/L ChemMapper web server, which is a hybrid 3D molecular DTT, 20 mmol/L MgCl2, 1 mmol/L phenylmethylsulfonyl similarity calculation approach designed to combine the fluoride, and 1 mg/mL lysozyme. Cell suspensions were strength of pharmacophore matching and volumetric overlay homogenized with a high-pressure cell disruptor (JN BIO) at that exhibits satisfactory "scaffold hopping" capability against 30,000 psi, and cell debris was removed by centrifugation at several representative kinases and has been utilized for ligand- 120,000 g for 40 minutes. Proteins were purified using a GST- based virtual screening (29). As PI stearoyl-arachidonyl acyl affinity column (GE Healthcare),andtheGSTtagwascleaved chains are long and flexible, a substituted compound with a overnight at 4 C with PreScission protease (GE Healthcare) in butyryl group attached to the oxygen atom of both PI side buffer containing 50 mmol/L HEPES (pH 7.5), 300 mmol/L chains was used. The structure of the substituted compound NaCl, 0.1% Triton X-100, and 2 mmol/L MgCl2. was drawn with ChemBio Office 2015, and MM2 minimization was used to obtain the starting structure for the similarity Kinases activity assay search. Using the substituted compound as the SHAFTS input The proteins used in this study were purchased from the molecule, the top 1,000 molecules with similarity scores > 1.0 following vendors: PI4KIIIa from Creative BioMart (catalog no. were reserved, resulting in 822 candidates. The crystal structure PI4KA-161H), PI3Ka from Invitrogen (catalog no. PV4788), of human PI4KIIa (PDB entry: 4HNE; ref. 26) was used as the PIK3Cd from Millipore (catalog no. 14-604-K), PIK3Cb from receptor for molecular docking. The docking accuracies of the Millipore (catalog no. 14-603-K), PIK3Cg from Invitrogen (cat- GLIDE 5.5 (Glide, version 5.5, Schrodinger,€ LLC), Gold 5.0 alog no. PR8641C), AKT1 from BPS (catalog no. 40003), AKT2

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from Carna (catalog no. 01-102), and AKT3 from Carna (catalog 30 minutes. The lysates were then divided into smaller (50 mL) no. 01-103, Lot. No. 05CBS-3274). In addition, PI4KIIa, PI4KIIb, aliquots and heated individually at different temperatures for and PI4KIIIb were expressed and purified as described above. The 3 minutes (S1000TM thermal cycler, Bio-Rad), followed by ADP-Glo kinase assay (Promega) was used to evaluate the kinase cooling for 3 minutes at room temperature. The appropriate activities of PI4KIIa, PI4KIIb, PI4KIIIa, PI4KIIIb, PI3Ka, PI3Kd, temperatures were determined in preliminary CETSA experi- PI3Kb, and PI3Kg according to previous reports with some mod- ments. The heated lysates were centrifuged at 20,000 g for 30 ifications (27). Briefly, 5 mL of kinase buffer containing different minutes at 4C to separate the soluble fractions from the concentrations of compounds (25 nmol/L PI4KIIa, 268 nmol/L precipitates. The supernatants were transferred to new micro- PI4KIIb, 83 nmol/L PI4KIIIa, 109 nmol/L PI4KIIIb, 1.65 nmol/L tubes and analyzed by SDS-PAGE followed by Western blot PI3Ka, 4.8 nmol/L PI3Kb, 7.6 nmol/L PI3Kg, or 17 nmol/L PI3Kd) analysis. was added to each well of the assay plate. For the PI4K assay, the fi buffer contained 50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, Detection and quanti cation of cellular PI(4)P, LPA, PI3P, PI(4,5)P , PI(3,4,5)P 0.1% anzergent 3–14, 1 mmol/L EDTA, and 20 mmol/L MgCl2, 2 3 while the buffer for the PI3K assay contained 50 mmol/L HEPES All the cellular lipid contents were measured using the appropriate Mass ELISA kits (Echelon Biosciences Inc.) according to the (pH 7.5), 3 mmol/L MgCl2, 1 mmol/L EGTA, 100 mmol/L NaCl, 0.03% CHAPS, and 2 mmol/L DTT. The kinase reaction was manufacturer's instructions. The kits used included the PI(4)P initiated by adding 5 mL of the substrate solution (50 mmol/L Mass ELISA Kit (catalog no. K4000E), the LPA Mass ELISA Kit (catalog no. K-2800S), the PI3P Mass ELISA Kit (catalog no. K- PI(4,5)P2 and 25 mmol/L ATP for the PI3Ks, 500 mmol/L PI, and 25 mmol/L ATP for the PI4Ks). The assay plates were then 3300), the PI(4,5)P2 Mass ELISA Kit (catalog no. K-4500), and the fl covered and incubated at room temperature for 1 hour, and the PI(3,4,5)P3 Mass ELISA Kit (catalog no. K-2500S). Brie y, cellular reactions were stopped by adding 10 mL of ADP-Glo reagent lipid extracts were analyzed for PI(4)P, LPA, PI3P, PI(4,5)P2 or PI (Promega). After equilibration for 40 minutes, 20 mL of kinase (3,4,5)P3 using the appropriate Mass ELISA Kit described above detection reagent (Promega) was added, and the mixture (40 mL and normalized by the protein concentration in the cell lysate. For total) was incubated for an additional 60 minutes before reading measurement of PI4P in tumor tissues, lipids extracted from equal the luminescence on a Varioskan Flash plate reader (Thermo weights (approximately 10 mg) of xenograft tumors were ana- Scientific). lyzed for PI4P using the PI4P Mass ELISA Kit described above and The kinase activities of AKT1, AKT2, and AKT3 were mea- normalized by the tissue weights. Statistical analysis was per- sured by a mobility shift assay. Briefly, 15 mL of kinase buffer formed with 6 paired cases (37). [50 mmol/L HEPES (pH 7.5), 0.0015% Brij-35] containing different concentrations of the compounds (2.5 nmol/L AKT1, Surface plasmon resonance–based binding assay 0.5 nmol/L AKT2 or 0.7 nmol/L AKT3) was added to each well The surface plasmon resonance (SPR) binding assays were of the assay plate. Then, a mixture consisting of 10 mLofFAM- performed on a Biacore T200 instrument (GE Healthcare) at labeled peptide (GL Biochem, 3 mmol/L) and ATP (60 mmol/L, 25 C. The PI4KIIa protein was covalently immobilized on a CM5 188 mmol/L, or 67 mmol/L)wasaddedtotheassayplate,which chip using a standard amine-coupling procedure in 10 mmol/L was then incubated at room temperature for 30 minutes. sodium acetate (pH 5.0). The chip was first equilibrated with HBS- Finally, the reaction was stopped by adding 25 mLofstop EP buffer [10 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 3 buffer [100 mmol/L HEPES (pH 7.5), 0.015% Brij-35, 0.2% mmol/L EDTA, 0.05% (v/v) surfactant P20, 0.1%(v/v) DMSO] coating reagent, 50 mmol/L EDTA], and the data were collected overnight. The compounds were serially diluted in HBS-EP buffer with the Caliper instrument (Perkin Elmer). and injected for 120 seconds (contact phase), followed by 120 seconds for the dissociation phase. The Kd values of the tested Thermal shift assay and cellular thermal shift assay compounds were determined using the Biacore T200 evaluation The thermal shift assay was performed on a 7500 Fast Real- software (GE Healthcare). Time PCR system (Applied Biosystems). Each reaction solution contained 5 mmol/L PI4KIIa,5 SYPRO orange (Sigma-Aldrich) Mouse xenograft experiments and the test compounds in 20 mL of buffer [50 mmol/L HEPES Eight-week-old male BALB/c nude mice (Weitonglihua) were fi (pH 7.5), 150 mmol/L NaCl, 2 mmol/L MgCl2], which was heated allowed to acclimate for 1 week under speci c pathogen-free from 25Cto95C at a 1% ramp rate. The melting temperature conditions in the animal facility of the Institute of Biophysics at (Tm) was calculated by the Boltzmann fitting method using the Chinese Academy of Sciences (Beijing, China). The mice were Protein Thermal Shift Software Version 1.1 (Applied Biosystems). subcutaneously injected with an MCF-7 cell suspension in a Each reaction was repeated three times. mixture of Matrigel and DMEM, and treatment was initiated after The cellular thermal shift assay (CETSA) was carried out 2 or 4 days. PI-273 was reconstituted in DMSO and administered based on a previously described protocol with minor modifica- by intraperitoneal injection. Tumor volume was monitored via tions (35, 36). For the in vivo assay, MCF-7 cells were harvested digital calipers three times each week and calculated using the and washed with PBS after the indicated treatments. All buffers formula (smallest diameter2 largest diameter)/2. All procedures were supplemented with complete protease inhibitor cocktail, involving animals and their care were approved by the animal and the cell suspensions were freeze-thawed three times using ethics committee of the Institute of Biophysics at the Chinese liquid nitrogen. The soluble fraction (lysate) was separated Academy of Sciences (Beijing, China). from the cell debris by centrifugation at 20,000 g for 30 minutes at 4C. For the in vitro assay, the cell lysates were Pharmacokinetics divided into two aliquots; one aliquot was treated with PI-273, PI-273 was formulated in a 0.9% saline solution containing and the other was treated with the diluent DMSO (control) for 10% DMSO and 0.9% Tween-80. Three male Sprague-Dawley

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(SD) rats were intravenously treated with 3.33 ml/kg formulated Statistical analysis PI-273, and another three were intragastrically treated with Statistical analyses were performed using two-tailed paired 10 mL/kg formulated PI-273. Blood samples were collected at Student t test. Differences were considered statistically significant 0.08, 0.16, 0.33, 0.67, 1, 1.5, 2, 3, and 5 hours after the intrave- when the P value was <0.01, as indicated in the legends. All data nous and intragastrical administrations. One 0.2-mL blood sam- are expressed as the means SD. ple was collected from each rat by retro-orbital bleeding. Blood was collected into heparin-containing tubes, and plasma was Results obtained by centrifugation at 4,000 rpm for 10 minutes. All Identification of PI-273 as a PI4KIIa inhibitor based on virtual protocols were based on standard operating procedures approved screening by the Institutional Animal Care and Use Committee (IACUC) In this study, virtual screening was employed to identify prom- of the Shanghai Institute of Materia Medica at the Chinese ising hits (Fig. 1A). The SPECS database (https://www.specs.net), Academy of Sciences (Beijing, China). The plasma concentrations containing 199,970 compounds, was used as the ligand database, of PI-273 were quantitated by the LC/MS-MS method, and non- and compounds with unfavorable physicochemical properties compartmental analysis using Phoenix 1.3 (Pharsight) was per- were filtered using Pipeline Pilot 7.5. The remaining 89,467 formed for all analytic measurements. The trapezoidal method compounds were used in a SHAFTS-3D ligand similarity search was used to calculate the area under the concentration–time curve with a PI-substituted compound as the input molecule, resulting (AUC), where AUC – ¼ AUC ¼ þ Ct/ke (ke is the elimination 0 ¥ 0- t in 822 candidates. The 822 compounds were docked into the rate constant). The mean residence time [(MRT) ¼ AUMC/AUC] PI4KIIa protein pocket near the palmitoylation insertion using and the elimination half-life [(t ) ¼ 0.693/ke] were also 1/2 the GLIDE 5.5 program. On the basis of the G-score and structural calculated. clustering, 142 structurally diverse compounds were selected and

Figure 1. Identiﬁcation of PI4KIIa small-molecule inhibitors. A, Schematic representation of the virtual screening strategy adopted in this study. B, Chemical structures of PI-273 and PI-69. C, The effect of PI-273 on cellular PI4P content. MCF-7 cells were treated with DMSO or the indicated concentrations of PI-273 for the indicated times. The cells were then harvested for PI4P measurement. D, MCF-7 cells were treated with DMSO or 1 mmol/L PI-273 for 24 hours before being collected for the indicated lipids analyses. The values are presented as the means SD from three independent experiments, and all the above experiments were performed three times in triplicate. , P < 0.01.

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purchased for biochemical assays. We then tested the effects of reaching a plateau. This effect was also observed in samples these compounds on PI4KIIa activity at 100 mmol/L and identi- treated with PI-93, PI-277, and PI-294, whereas PI-69 did not fied 7 that exhibited greater than 60% kinase activity inhibition affect the PI4KIIa Tmvalue (Fig. 2B; Supplementary Fig. S3B), (Supplementary Fig. S1A). Measuring the half-maximal inhibi- consistent with their individual potencies for PI4KIIa inhibition. tory concentration (IC50) values of these compounds for kinase To evaluate the in vivo interaction between PI-273 and PI4KIIa,we activity in vitro (Supplementary Fig. S1B) and cell viability in vivo conducted a CETSA (35). The CETSA, developed by D. Molina (Supplementary Fig. S1C) revealed that PI-93 (Supplementary Martinez and his colleagues, is used for assessing target engage- Fig. S1D) exhibited the strongest inhibitory effect. We then further ment by drugs in vivo. The CETSA is based on the biophysical tested the effect of PI-93 on the intracellular lipid content. As principle of the ligand-induced thermal stabilization of target shown in Supplementary Fig. S1E, the MCF-7 cells incubated with proteins, and it will likely become a valuable tool for validating 20 mmol/L PI-93 for 24 hours exhibited lower PI4P levels than and optimizing drug target engagement (35, 36). As shown in Fig. DMSO-treated cells, while the LPA, PI3P, PI(4,5)P2, and PI(3,4,5) 2C, intact MCF-7 cells treated with 1 mmol/L PI-273 for 96 hours P3 levels were not significantly different in either sample. and cell lysate treated with 20 mmol/L PI-273 for 30 minutes On the basis of the SHAFTS-3D ligand similarity search, anoth- exhibited shifted melting curves for PI4KIIa. Under DMSO treat- er 53 candidates were selected and purchased for the second ment, 50% of PI4KIIa was degraded at 60C, whereas in the PI- round of screening. Six compounds with an inhibitory effect on 273–treated samples, 50% degradation required temperatures of PI4KIIa activity stronger than that of PI-93 were selected for 70C (intact cells) and 75C (cell lysate). Thus, PI-273 inhibits further analysis (Supplementary Fig. S2A; Fig. 1A). The chemical PI4KIIa activity via a direct binding event. We then assessed the structures of these representative PI-93 analogues are presented in reversibility of the binding between PI-273 and PI4KIIa. PI4KIIa Supplementary Table S1 (in addition to the 6 selected com- was preincubated with varying concentrations of PI-273 (0–1 pounds, 5 compounds that shared a common parent nucleus mmol/L) for different lengths of time (0–60 minutes) before the with PI-93 in the first-round screening, and another 3 compounds kinase reaction was initiated. Only the concentration of PI-273 that had an inhibition effect similar to that of PI-93 in the second- and not the incubation time influenced the inhibitory effect of PI- round screening have also been included) and Fig. 1B. The 273 on PI4KIIa activity (Fig. 2D), suggesting that the interaction superimposition of PI with these six representative active com- between PI-273 and PI4KIIa is reversible. As further confirmation, pounds is shown in Supplementary Fig. S2B. The IC50 values of we evaluated the enzyme kinetics of PI4KIIa, which revealed that the compounds of interest for PI4KIIa kinase activity in vitro the slope of the velocity-to-enzyme concentration decreased with and cell viability in vivo in MCF-7 cells are presented in Supple- increasing PI-273 concentration (Fig. 2E), thus indicating that PI- mentary Table S1. 2-(3-(4-chlorobenzoyl)thioureido)-4-ethyl-5- 273 is a reversible inhibitor of PI4KIIa. Taken together, these methylthiophene-3-carboxamide (PI-273) had the greatest inhib- results strongly indicate that PI-273 directly interacts with PI4KIIa itory effect of MCF-7 cell viability, and its IC50 value in the in a reversible manner. biochemical assay was 0.47 mmol/L. In addition, our results (Supplementary Fig. S2C) also indicated that PI-273 had the best PI-273 is a PI-competitive inhibitor of PI4KIIa inhibitory effect on the cellular PI4P content among the analogues There are three types of reversible inhibitors: competitive, (PI-294, PI295, PI-308), which was consistent with the analogues uncompetitive, and anticompetitive. As described above, PI- effects on cell viability and AKT activity. Other PI-273 analogues, 273 is a reversible inhibitor of PI4KIIa, and enzyme kinetic such as PI-69/107/114, that had no effect on PI4KIIa activity also analysis was performed to determine the exact type of this had no effect on MCF-7 cells (Supplementary Table S1). In the inhibition (Fig. 3A and B). At varying concentrations of ATP, PI4P content assay, PI-273 reduced the intracellular PI4P content increasing PI-273 decreased the maximum velocity (Vmax) of the fi – in MCF-7 cells in a dose- and time-dependent manner (Fig. 1C). reaction (Fig. 3A and C), but the apparent af nity (KM, Michaelis Furthermore, as shown in Fig. 1D, PI-273 treatment could spe- Menten constant) of ATP for the binding site did not change (Fig. cifically inhibit PI4P production but not that of other lipids, such 3A and D), indicating that PI-273 is an ATP-uncompetitive as LPA, PI3P, PI(4,5)P2 and PI(3,4,5)P3. Taken together, these inhibitor. However, when we varied the concentration of PI, the results suggest that PI-273 is a structurally novel PI4KIIa inhibitor Vmax of the reaction remained unchanged (Fig. 3B and C), whereas with remarkable potency against the kinase activity of PI4KIIa. the KM value for PI at the binding site increased, indicating that PI- 273 is a PI-competitive inhibitor (Fig. 3B and D). We next PI-273 directly binds and reversibly inhibits PI4KIIa performed experiments with PI4KIIa mutants to verify this con- To validate these potential PI4KIIa inhibitors, direct interac- clusion (Fig. 3E; Supplementary Table S2). As expected, the Tm tions between PI4KIIa and the candidate compounds were mea- of wild-type PI4KIIa was significantly elevated in the presence of sured by SPR. As shown in Fig. 2A and Supplementary Fig. S3A, the PI-273. However, mutations or deletions in the PI-binding reg- interactions were dose-dependent. The equilibrium dissociation ulation region (for example, the palmitoylation insertion, D165- constant (Kd) between PI-273 and PI4KIIa was approximately 173, or K165E/K168E/K172E), but not those at the nucleotide- 9.49 mmol/L based on the association rate constant (kon¼ 2.08 binding site (for example, K152A, D346A), reduced the DTm 2 1 1 10 M s ) and the dissociation rate constant (koff ¼ 2.13 value. We previously reported that the palmitoylation insertion 3 1 10 s ), whereas the Kd of PI-277 was 9.59 mmol/L and the Kd of modulates kinase activity by tuning the PI-binding pocket (26, PI-282 was 5.91 mmol/L (these two compounds have the best 38), and these results confirm that PI-273 is a PI-competitive solubility among PI-273/274/275/276/282/294/295/308; listed inhibitor of PI4KIIa. in Supplementary Table S1). The direct interaction between these compounds and PI4KIIa was further confirmed by a fluorescence- PI4KIIa is required for the inhibitory effect of PI-273 based thermal shift assay; PI-273 dose dependently shifted the The selectivity of kinase inhibitors is crucial to avoid side effects melting temperature (Tm) of PI4KIIa by more than 4 C before caused by off-target inhibition (39). To validate the subtype-

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Figure 2. PI-273 directly binds and reversibly inhibits PI4KIIa. A, Representative SPR binding curves for PI4KIIa and 2-fold serial dilutions of PI-273 from 1.25 mmol/L to 20 mmol/L. B, Thermal shift assay indicating the stabilization of PI4KIIa by PI-273. Melting curves for 5 mmol/L PI4KIIa protein in the presence of varying concentrations of PI-273 or PI-69. C, A cellular thermal shift assay was performed to evaluate the interaction between PI-273 and PI4KIIa in intact cells (in vivo)and cell lysates (in vitro). D, PI4KIIa was preincubated with the indicated concentrations of PI-273 (0–1 mmol/L) for different durations (0–60 minutes) before ATP was added. The graphs represent normalized percentage of inhibition compared with preincubation time. E, The initial velocity kinetics compared with varying PI4KIIa enzyme concentrations (2.5–10 ng/mL) at the indicated concentrations of PI-273 (0–2 mmol/L). All the above experiments were performed three times with comparable results.

speciﬁc inhibitory effect of PI-273 on PI4KIIa, we tested the our previous results. The PI-273 IC50 values were much higher for inhibitory effects of PI-273 on 11 related kinases, including four the other kinases than for PI4KIIa, even for the isoform having the PI4K subtypes (PI4KIIa, PI4KIIb, PI4KIIIa, and PI4KIIIb), four greatest homology to PI4KIIa, PI4KIIb, which had a PI-273 IC50 PI3K subtypes (PI3Ka, PI3Kb, PI3Kg, PI3Kd) and three AKT value more than 30-fold higher than that for PI4KIIa. For the subtypes (AKT1, AKT2, and AKT3). As shown in Fig. 4A, the IC50 other PI4K subtypes (PI4KIIIa and PI4KIIIb), whose structures are values of PI-273 for PI4KIIa were 0.47 mmol/L, consistent with more similar to those of PI3Ks, the IC50 values were both more

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Figure 3. PI-273 is a substrate-competitive inhibitor of PI4KIIa. Enzyme kinetics analysis of PI4KIIa inhibition by PI-273. Activity assays were performed with varying concentrations of ATP (A) and PI (B). Double-reciprocal plots of initial velocities (Lineweaver–Burk plots) showing uncompetitive inhibition by PI-273 toward ATP

and purely competitive inhibition toward PI as a substrate. The experiment was performed three times with comparable results. Vmax (C)andKM (D) values for ATP and PI under varying concentrations of PI-273. E, Thermal shift assay demonstrating the stabilization of PI4KIIa variants by PI-273. PI4KIIa (5 mmol/L) variants were treated with PI-273 (50 mmol/L) and then subjected to the thermal shift analysis. The values are presented as the means SD from three independent experiments (Student t test), and this experiment was performed three times in triplicate. , P < 0.01; #ns, not signiﬁcant.

than 100 mmol/L. Although the PI-273 IC50 value for PI3Kb was staining (Supplementary Fig. S5C). In the cell viability assay, the only 10-fold higher than that for PI4KIIa, our results shown in Fig. PI4KIIa knockout greatly decreased the sensitivity to PI-273. 1D indicate that PI-273 treatment cannot reduce PI3P or PI(3,4,5) Compared with the DMSO-treated wild-type MCF-7 cells, 10 P3 content, suggesting that PI3Kb is not a major target of PI-273 in mmol/L PI-273 could reduce the cell viability by approximately vivo. All the above results indicate that PI-273 is highly specific for 80%, while this ratio for the PI4KIIa knockout MCF-7 cells was PI4KIIa. only 25% (Fig. 4B), indicating that PI4KIIa is essential for the We next tested the specificity of PI-273 for PI4KIIa in vivo.We effect of PI-273 on cell viability. Because our previous works (4, carried out genome-scale CRISPR-Cas9 knockout screening for PI- 17) and other studies indicated that PI4KIIa can regulate the AKT 273-resistant genes (Supplementary Fig. S4A). Supplementary signaling pathway (3), we tested the effects of our compounds on Figure S4B shows that PI4KIIa is the most significantly enriched AKT signaling. As shown in Fig. 4C and Supplementary Fig. S6A, target, whose function loss can confer strong protection to MCF-7 PI-273 could suppress the AKT signaling pathway in a dose- and cells against PI-273. We then generated PI4KIIa knockout MCF-7 time-dependent manner. However, other analogues, such as PI- cells using the CRISPR-Cas9 method (Supplementary Fig. S5). As 294 (Supplementary Fig. S6B), PI-250 (Supplementary Fig. S6C), expected, obtaining MCF-7 cells in which PI4KIIa was totally PI-295, and PI-308 (Supplementary Fig. S6D) had much weaker knocked out proved to be difficult. Among 200 individual mono- effects on this signaling pathway, consistent with their effects on clones, only one PI4KIIa knockout cell line was selected, and its cell viability and PI4P content. These results indicate that PI4KIIa sequencing results indicated that PI4KIIa exists as a multicopy is the target for PI-273. We next addressed whether PI4KIIa is gene in MCF-7 cells (Supplementary Fig. S5A). We then further solely essential for the effects of PI-273 in vivo using PI4KIIa confirmed the knockout effects of this clone by Western blot knockout cells, and we found that PI-273 reduced the PI4P analysis (Supplementary Fig. S5B) and immunofluorescence content by 40% in wild-type (CTR) cells but had no effect on

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Figure 4.

PI-273 inhibits PI4KIIa downstream signaling transduction in breast cancer cells. A, PI-273 IC50 values for PI4KIIa, PI4KIIb, PI4KIIIa, PI4KIIIb, PI3Ka, PI3Kd, PI3Kb, PI3Kg, AKT1, AKT2, and AKT3. B, The antiproliferation activity of PI-273 in wild-type MCF-7 cells and PI4KIIa knockout MCF-7 cells. C, Speciﬁc regulation of AKT signaling by PI-273 in MCF-7 cells. MCF-7 cells were treated with the indicated doses of PI-273 for 3 days, followed by treatment with 100 ng/mL EGF

for 10 minutes. AKT phosphorylation and protein expression levels were measured by Western blot analysis. The effects of PI-273 on LPA, PI4P, PI3P, PI(4,5)P2, PI (3,4,5)P3 content (D) and AKT signaling in wild-type MCF-7 cells (CTR cells) and PI4KIIa knockout MCF-7 cells (KO cells; E). Cells were treated with 1 mmol/L PI-273 or DMSO for 24 hours (for lipid ratio detection) or 3 days (for AKT signaling detection), respectively. Lipid contents were detected by the appropriate Mass ELISA Kit. AKT phosphorylation and protein expression levels were measured by Western blot analysis. All the values are presented as the means SD from three independent experiments, and all the above experiments were performed three times in triplicate. , P < 0.01; #ns, not signiﬁcant.

PI4P in PI4KIIa knockout (KO) cells. In contrast, PI-273 had no Effect of PI-273 on breast cancer cells effect on other lipid [LPA, PI3P, PI(4,5)P2 or PI(3,4,5)P3] content Compounds PI-273, PI-274, PI-277, PI-294, PI-295, and PI- in wild-type cells or PI4KIIa knockout cells (Fig. 4D). Consistent 308 inhibited PI4KIIa activity both in vitro and in vivo and had with this result, PI-273 decreased the p-AKT levels by 50% in wild- varying effects on the viability of MCF-7 cells. PI-273 had the type cells but had no effect on their phosphorylated levels in greatest effect on PI4KIIa inhibition and was thus selected PI4KIIa knockout cells (Fig. 4E). In addition, PI-273 did not affect for further exploration. To examine the biological effects of the PI4P level (Supplementary Fig. S7A) or the AKT signaling PI4KIIa inhibition in greater detail, we tested the inhibitory effect pathway (Supplementary Fig. S7B) in the PI4KIIa knockdown of PI-273 on different breast cancer cell lines: MCF-7, T-47D, SK- cells generated by siRNA. Taken together, the in vitro and in vivo BR-3, BT-474, MDA-MB-468, MDA-MB-231 (KRas mutant), experimental results indicate that PI-273 is a PI4KIIa-speciﬁc SUM229PE (KRas mutant), SUM159PT (HRas mutant), and Hs inhibitor. 578T (HRas mutant; ref. 40). The characteristics of the cell lines

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Figure 5. PI-273 inhibits the proliferation of breast cancer cells. A, Cell viability assays (WST-8 assay) were performed in MCF-7, T-47D, SK-BR-3, BT-474, MDA-MB-468, MDA- MB-231, SUM229PE, SUM159PT, and Hs 578T cells after treatment with the indicated concentrations of PI-273 for 72 hours. B, Six breast cancer cells were treated for 48 hours with DMSO or 2 mmol/L PI-273 and then labeled with PI. Cells were analyzed by ﬂow cytometry. The graph shows the percentage of cells in each cell-cycle phase. C, Apoptosis ratio in cells treated with PI-273. Cells were counted using a TUNEL kit after being treated with DMSO or 2 mmol/L PI-273 for 48 hours. The percentage of apoptotic cells are presented on the y-axis. D and E, Effects of PI-273 on colony formation in MCF-7, T-47D, Hs 578T, and MDA-MB-231 cells. Anchorage-dependent cell growth and anchorage-independent cell growth were measured using the plate clone-forming assay and the soft agar clone-forming assay, respectively. Colony numbers were determined after 7 days of incubation for the plate clone-forming assay and 14 days for the soft agar clone- forming assay. Scale bar, 200 mm. All the values are presented as the means SD from three independent experiments (Student t test), and all the above experiments were performed three times in triplicate. , P < 0.01; #ns, not signiﬁcant.

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used in this study, including the statuses of the primary tumor, intraperitoneal injection of PI-273 at 50 mg/kg/2 days also origin, estrogen receptor (ER), progesterone receptor (PR), human inhibited MCF-7 xenograft tumor growth, although with lower epidermal growth factor receptor 2 (HER-2), Ras, mice tumori- efficiency than the 25 mg/kg/day injection (Supplementary Fig. genicity and the PI-273 IC50 value, are summarized in Supple- S9A and S9B). We also assessed the toxic effects of PI-273 in mice, mentary Table S3. As shown in Fig. 5A, Ras-mutant breast cancer and no lethargy, weight loss (Fig. 6C), or other physical indicators cell lines are less sensitive to PI-273, as the PI-273 IC50 values for of sickness were observed. Various tissues, including liver, intes- MCF-7, T-47D, SK-BR-3, MDA-MB-468, and BT-474 cells were tine, lung, kidney, spleen and stomach tissue, were examined by 3.5 mmol/L, 3.1 mmol/L, 2.3 mmol/L, 3.9 mmol/L and 2.1 mmol/L, hematoxylin eosin (H&E) staining, and no tissue damage (mac- respectively, while the IC50 values for all the Ras-mutant cell lines roscopic or microscopic) was observed (Supplementary Fig. S9C). were higher than 10 mmol/L. The 56-carboxyfluorescein diacetate These studies establish the effectiveness and safety of PI-273 for succinimidyl ester (CFDA SE) assay was used to evaluate the antitumor applications. We further assessed the utility of PI-273 effects of PI-273 on cell proliferation. As shown in Supplementary in animal experiments. SD rats were treated with 0.5 mg/kg PI-273 Fig. S8A, 1 mmol/L and 2 mmol/L PI-273 inhibited the cell intravenously or 1.5 mg/kg PI-273 intragastrically. Blood samples proliferation of both MCF-7 and T-47D cells in a time-dependent collected retro-orbitally were collected at 0.08, 0.16, 0.33, 0.67, 1, manner, but these PI-273 doses did not influence the proliferation 1.5, 2, 3, and 5 hours after the intravenous and intragastrical of Hs 578T cells. These results are consistent with the inhibition of administrations. The plasma levels were analyzed by LC/MS-MS, these compounds against PI4KIIa in different cell lines and and the summarized pharmacokinetic results are presented indicate that MCF-7 and T-47D cells are much more sensitive to in Table 1. Compound PI-273 is retained in rats at a half-life of PI-273. Cell-cycle regulation by PI-273 was examined by 3,8- 0.411 hours for intravenous administration and 1.321 hours for diamino-5-[3-(diethylmethylammonio) propyl]-6-phenylphe- intragastrical administration, and the absolute bioavailability of nanthridinium diiodide staining and flow cytometry. Interesting- PI-273 is 5.1%. These pharmacokinetic results indicate that PI- ly, PI-273 blocked the cell cycle at the G2–M phase (Fig. 5B), 273 as lead compound showed moderate pharmacokinetic activ- similar to the effect of PIK-75, another substrate-competitive ities. Histologic examination using the TUNEL assay to detect inhibitor of PIKs (PI3Ka; ref. 41). The effect of PI-273 on cell apoptotic cells revealed a significant increase in the number of apoptosis was evaluated by a terminal deoxynucleotidyl transfer- apoptotic cells in the PI-273–treated MCF-7 xenografts compared ase dip nick end labeling (TUNEL) assay. As shown in Fig. 5C, with those treated with vehicle (Fig. 6D). A significant decrease in treatment with PI-273 induced cell apoptosis in all three Ras wild- proliferation, measured by Ki-67 staining, was observed in the PI- type breast cancer cells: MCF-7, T-47D, and SK-BR-3. In addition 273–treated MCF-7 xenografts compared with the vehicle to the cell viability assay, the inhibitory effects of PI-273 on the (Fig. 6E). The PI4P content and p-AKT levels in MCF-7–induced tumorigenicity of cells in vitro were evaluated with plate clone- tumors were measured to evaluate the effect of PI-273 in the forming tests (Fig. 5D) and soft agar clone-forming tests (Fig. 5E). xenograft model. Consistent with the data shown above, the PI- PI-273 exhibited prominent drug potency in MCF-7 and T-47D 273–treated tumors exhibited reduced PI4P content and p-AKT cells (1 mmol/L PI-273 reduced both plate colony and soft agar levels (Fig. 6F). These results confirm that the PI4KIIa-specific SMI colony growth by more than 50%). However, Hs 578T and MDA- PI-273 suppresses proliferation, survival, and AKT signaling in MB-231 cells were less sensitive to PI-273 than MCF-7 and T-47D MCF-7–induced breast cancer in vivo. The mechanism underlying cells. To assess whether sensitivity is dependent on PI4KIIa the PI-273 antitumor effect is summarized in Fig. 6G; PI-273 expression in different cell lines, the PI4KIIa expression levels inhibits the kinase activity of PI4KIIa, leading to reduction of the (Supplementary Fig. S8B) and PI4P regulation effects (Supple- intracellular PI4P content and suppression of the PI3K/AKT mentary Fig. S8C) were measured. The results showed that all the signaling pathways, resulting in retarded cell proliferation and cell lines had high PI4KIIa expression levels, and the PI4P content increased apoptosis, and, consequently, tumor growth inhibition. could be regulated by PI-273 in all four cell lines tested. Although PI-273 shows good potential for use in clinical trials for human the PI4KIIa expression levels were different among these cell lines, breast cancer. such differences could not distinguish the sensitivity of PI-273. However, the cell sensitivities to PI-273 showed strong correlation Discussion with the Ras status (Fig. 5A–E; Supplementary Table S3). Taken together, these results indicate that PI-273 retards cell prolifera- Because PI4KIIa plays important roles in Golgi trafficking (14, tion by blocking cells at the G2–M phase, inducing cell apoptosis 42, 43) and tumor progression (3, 4, 44), SMIs of PI4KIIa have and suppressing cell tumorigenicity, and these effects are depen- potential as chemical tools for studying the biological function of dent on the cell type. PI4KIIa and breast cancer treatment (10, 19, 42). Here, PI-273 was screened out and identified as the most potent inhibitor of Antitumor effect of PI-273 in a xenograft model PI4KIIa activity and breast cancer cell viability. Biochemistry and To determine whether PI-273 can suppress breast cancer in vivo, kinetic analyses revealed that PI-273 directly binds and reversibly we engrafted MCF-7 cells, which were confirmed to be sensitive to inhibits PI4KIIa. The fact that PI-273 is a substrate-competitive PI-273 in the above experiments, into the right flank region of inhibitor rather than an ATP-competitive inhibitor is its greatest BALB/c nude mice. Four days after cell injection, the mice were advantage, as this results in kinase isoform selectivity. Further randomized and received either an intraperitoneal injection of PI- study confirmed that PI4KIIa is essential for the effects of PI-273. 273 25 mg/kg/day or vehicle (5% DMSO). The mice were sacri- As the first substrate-competitive inhibitor in the PI4K family and ficed 24 hours after the 15th injection. PI-273 profoundly sup- the third such inhibitor in the entire PIK family [PIK-75 is a pressed the tumor volume (Fig. 6A) and weight (Fig. 6B) in the substrate-competitive inhibitor of PI3K (23) and NIH-12848 is a MCF-7 xenografts compared to the effects of treatment with the substrate-competitive inhibitor of PI5P4Kg (25)], PI-273 is highly vehicle. Another group of parallel experiments indicated that an isoform-selective and has significant anti-breast cancer effects

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Figure 6. PI-273 suppresses breast cancer in vivo. Tumor growth curve (A), tumor weight (B), and mouse weight (C) for MCF-7 xenografts in BALB/c nude mice treated with 25 mg/kg/day PI-273 or an equal volume of vehicle for 16 consecutive days. Statistical analysis was performed using the paired t test. D, TUNELþ staining of the histologic sections of MCF-7 xenografts. The y-axis represents the ratio of TUNELþ cells per ten fields for one tumor sample (n ¼ 5 tumors/treatment). Scale bar, 20 mm. E, Ki-67 IHC staining of xenograft MCF-7 tumor sections. The y-axis represents the ratio of Ki-67þ cells per ten fields for one tumor sample (n ¼ 5 tumors/treatment). Statistical analysis was performed using Student t test. Scale bar, 100 mm. F, Effect of PI-273 on the phosphorylation levels of AKT and the PI4P content in MCF-7 xenograft tumors. All the values are presented as the means SD from the indicated samples. , P < 0.01. G, Model of the antitumor effect of PI-273. PI-273 can specifically bind PI4KIIa, inhibiting its activity and, notably, reducing the cellular PI4P content, thus resulting in suppressed PI3K/AKT signaling transduction. This activity blocks cells at the G2–M phase, resulting in proliferation arrest and apoptosis. Together, these effects can retard the growth of breast tumors.

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Table 1. Pharmacokinetic results of the SD rats after intravenous administration structure, hydrogen deuterium exchange (HDX), and single-mol- of 0.5 mg/kg PI-273 and intragastrical administration of 1.5 mg/kg PI-273, ecule fluorescence resonance energy transfer (FRET) analysis. respectively. In this study, we demonstrated that PI-273 can inhibit breast PI-273 cancer cell proliferation (Supplementary Fig. S8A), block the cell Intravenously Intragastrically Pharmacokinetic (0.5 mg/kg) (1.5 mg/kg) cycle (Fig. 5B) and induce cell apoptosis (Fig. 5C). We then further parameters Mean SD Mean SD confirmed its effects on colony formation (Fig. 5D and E) and on 1 Cmax (ng*mL ) 2392.7 46 116 23 an MCF-7 cell-induced xenograft model (Fig. 6A and B), all of Tmax (h) 0.083 0.00 0.278 0.096 which indicated the suppressive effect of PI-273 on breast cancer t 1/2 (h) 0.411 0.087 1.321 0.657 growth both in vitro and in vivo. Because previous studies from our MRT0–12 (h) 0.23 0.072 1.25 0.061 group and others have suggested that in addition to breast cancer, MRT0–¥ (h) 0.23 0.072 1.571 0.378 1 1 many other cancers, such as malignant melanoma and thyroid CL/F (L*h *kg ) 0.649 0.204 11.4 1.32 1 Vd/F (L*kg ) 0.38 0.118 21.0 7.86 carcinoma, also feature high PI4KIIa expression (4, 16), we 1 AUC0–12 (ng*h*mL )816 218 125 8.28 suspect that PI-273 may have inhibitory effects on these types of 1 * * AUC0–¥ (ng h mL )816218 132 16.0 tumors. In our experiment, we also tested the effects of PI-273 on MDA-MB-435 cells, which have been identified as malignant melanoma cells (50–52). To our surprise, the MDA-MB-435 cells without toxicity, showing its great potential for basic PI4KIIa and were even more sensitive to PI-273 in both the plate clone- pharmaceutical research. forming test and the soft agar clone-forming test compared Numerous small molecules have been previously identified as with the MCF-7 and T-47D cells (data not shown), suggesting PIK inhibitors, as summarized in Supplementary Table S4, and that PI-273 may suppress melanoma growth and merits further none affect PI4KIIa, except for EGCG (45), resveratrol (46), and investigation. adenosine (47). In fact, we tested the effects of all the potential Because precision medicine is considered the optimal means candidates on PI4KIIa activity prior to screening, including of completely conquering cancer, clarifying which patient EGCG, resveratrol, adenosine, PI3K inhibitors (Wortmannin, categories are sensitive to particular drugs is important. In our LY294002, ZSTK474), a PI3Ka inhibitor (PI103), a PI3Kd inhib- study, Hs 578T, MDA-MB-231, SUM229PE, and SUM159PT itor (IC87114), a PI3Kg inhibitor (AS605240), and a PI4KIIIb cells were relatively resistant to PI-273 in terms of cell viability, inhibitor (PIK93; Supplementary Fig. S10). As observed previ- cell cycle, and apoptosis (Fig. 5), which is consistent with ously, PI4KIIa was insensitive to PI3K and type III PI4K inhibitors previous studies concluding that monotherapies targeting the (10, 48, 49), and the IC50 values of both resveratrol and EGCG PIK signaling pathway are largely disappointing for Ras- were greater than 100 mmol/L. Moreover, these molecules are all mutant cancers (53). PI3K/AKT and Ras/MEK/ERK are two broad-spectrum inhibitors and thus cannot be used for PI4KIIa crucial interlinked growth and survival signaling pathways in subtype-specific functional studies. Therefore, screening subtype- tumors (52), and Torbett and colleagues indicated that Hs specific inhibitors for PI4KIIa is of great significance. 578T cells with activated forms of the H-Ras oncogene result in As summarized in Supplementary Table S4, hundreds of small the activation of parallel pathways that are able to compensate molecules were identified as PIK inhibitors by screening, but most for the loss of PI3K signals. Different groups have indicated were ATP-competitive, except for PIK-75 (23) and NIH-12848 that PI3K inhibitor treatment alone does not result in the (25), limiting their potential for high isoform selectivity. The lack regression of Ras-mutant lung tumors (54, 55), pancreatic of structural information regarding the PIK substrate complex is a cancer (56), or non–small cell lung cancer (57) because of barrier to developing substrate-competitive inhibitors because Ras/MEK/ERK–dependent signaling activation. However, com- lipid substrate binding requires the kinase to adopt a mem- pared with monotherapies, combining the two treatments brane-bound conformation that is likely different from the struc- (MEK and PI3K inhibitors) resulted in a significant survival ture observed in crystal form. We have recently overcome advantage in these cancers. Taken together, these results sug- this limitation by combining data generated from crystal struc- gest that the pharmacodynamic effects of PI-273 may also be ture, molecular docking, and biochemical studies to identify related to Ras activity, which is another interesting topic to be the putative PI-binding pocket of PI4KIIa. We determined that explored in future work. a PI4KIIa-unique insertion, the palmitoylation insertion, Interestingly, because PI-273 treatment and PI4KIIa knockout affects the conformation of the substrate-binding pocket and thus had negligible effects on the PI(4,5)P2 and PI(3,4,5)P3 levels regulates its kinase activity (26). In this study, we found that PI- (Figs. 1D and 4D), we hypothesize that the effect of PI4KIIa 273 can directly bind the palmitoylation insertion and functions inhibitors on AKT activity does not result from the direct regu- as a PI-competitive reversible inhibitor (Figs. 2D, 2E, 3A, lation of the PI(4,5)P2 and PI(3,4,5)P3 levels. Instead, numerous and 3B; Table 1). These results also confirm our previous MD studies have indicated that PI4KIIa is important for receptor simulation results that the palmitoylation insertion influences activity via trafficking regulation (13, 17, 58, 59). Therefore, it substrate binding. Our work suggests that MD simulation may be is possible that the regulation of PI4KIIa inhibitors on AKT helpful for identifying PIK substrate-binding pockets, thus facil- activity is dependent on its receptor regulation effect, but this itating substrate-competitive inhibitor screening. However, with requires further validation. respect to the PI-competitive mechanism of PI-273, determining In summary, we identified PI-273 as a lead substrate-compet- whether PI-273 can directly bind the substrate pocket or only itive inhibitor of PI4KIIa based on a unique insertion, the pal- induce allosteric regulation of the substrate-binding site is diffi- mitoylation insertion, of PI4KIIa as a binding site. Our results also cult because of the lack of inhibitor-soaked crystal structure demonstrate the significance, safety, and efficacy of PI4KIIa as a information. Additional efforts to elucidate the binding mecha- therapeutic target, and PI-273 will be optimized as a therapeutic nism of PI-273 will be made in the future using NMR, crystal agent for cancer in the future.

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Disclosure of Potential Conflicts of Interest Grant Support No potential conflicts of interest were disclosed. This research was supported by National Key R&D Program of China (2017YFA0504000, 2016YFC0903100 to C. Chen), Personalized Medicines- Authors' Contributions Molecular Signature-based Drug Discovery and Development, the Strategic Conception and design: J. Li, Z. Gao, D. Zhao, H. Jiang, C. Luo, C. Chen Priority Research Program of the Chinese Academy of Sciences (XDA12020316 Development of methodology: J. Li, Z. Gao, L. Zhang, C. Luo to C. Chen), the National Natural Sciences Foundation of China (31570857 and Acquisition of data (provided animals, acquired and managed patients, 31225012 to C. Chen; 31101021 and 81472839 to J. Li; and 81430084, provided facilities, etc.): J. Li, Z. Gao, X. Qiao, Y. Zhao, P. Zhang 81625022, and 21472208 to C. Luo), the "863" National High-Technology Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Development Program of China (0A200202D03 to C. Chen), National computational analysis): J. Li, Z. Gao, D. Zhao, Y. Zhao, H. Ding, J. Lu, H. Jiang, Key Scientific Instrument & Equipment Development Program of China C. Luo, C. Chen (2012YQ03026010 to C. Luo), NNCAS-2012-2 to C. Chen, the Beijing Writing, review, and/or revision of the manuscript: J. Li, Z. Gao, D. Zhao, Natural Science Foundation (7132156 to J. Li), and the Science and Technology C. Luo, C. Chen Commission of Shanghai Municipality (15431903100 to J. Li) Administrative, technical, or material support (i.e., reporting or organizing The costs of publication of this article were defrayed in part by the data, constructing databases): J. Li, Z. Gao, D. Zhao, L. Zhang, J. Liu, C. Chen payment of page charges. This article must therefore be hereby marked Study supervision: H. Jiang, C. Luo, C. Chen advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Acknowledgments We thank Pietro De Camilli for providing the PI4KIIa antibody and Shane Received February 22, 2017; revised June 29, 2017; accepted August 14, 2017; Minogue for providing the full-length human PI4KIIa cDNA. published OnlineFirst August 21, 2017.

References 1. Di Paolo G, De Camilli P. Phosphoinositides in cell regulation and onset degeneration of spinal cord axons. Proc Natl Acad Sci U S A membrane dynamics. Nature 2006;443:651–7. 2009;106:11535–9. 2. Ree AH, Flatmark K, Saelen MG, Folkvord S, Dueland S, Geisler J, 16. Waugh MG.Phosphatidylinositol 4-kinases, phosphatidylinositol 4-phos- et al. Tumor phosphatidylinositol 3-kinase signaling in therapy phate and cancer. Cancer Lett 2012;325:125–31. resistance and metastatic dissemination of rectal cancer: opportu- 17. Li J, Zhang L, Gao Z, Kang H, Rong G, Zhang X, et al. Dual inhibition of nities for signaling-adapted therapies. Crit Rev Oncol Hematol EGFR at protein and activity level via combinatorial blocking of PI4KIIal- 2015;95:114–24. pha as anti-tumor strategy. Protein Cell 2014;5:457–68. 3. Chu KM, Minogue S, Hsuan JJ, Waugh MG. Differential effects of the 18. Tokuda E, Itoh T, Hasegawa J, Ijuin T, Takeuchi Y, Irino Y, et al. Phospha- phosphatidylinositol 4-kinases, PI4KIIalpha and PI4KIIIbeta, on Akt acti- tidylinositol 4-phosphate in the Golgi apparatus regulates cell-cell adhe- vation and apoptosis. Cell Death Dis 2010;1:e106. sion and invasive cell migration in human breast cancer. Cancer Res 4. Li J, Lu Y, Zhang J, Kang H, Qin Z, Chen C. PI4KIIalpha is a novel regulator 2014;74:3054–66. of tumor growth by its action on angiogenesis and HIF-1alpha regulation. 19. Waugh MG. Amplification of chromosome 1q genes encoding the phos- Oncogene 2010;29:2550–9. phoinositide signalling enzymes PI4KB, AKT3, PIP5K1A and PI3KC2B in 5. McNamara CW, Lee MC, Lim CS, Lim SH, Roland J, Nagle A, et al. Targeting breast cancer. J Cancer 2014;5:790–6. plasmodium PI(4)K to eliminate malaria. Nature 2013;504:248–53. 20. Pan W, Choi SC, Wang H, Qin Y, Volpicelli-Daley L, Swan L, et al. Wnt3a- 6. Bothmer J, Markerink M, Jolles J. Evidence for a selective decrease in type 1 mediated formation of phosphatidylinositol 4,5-bisphosphate regulates phosphatidylinositol kinase activity in brains of patients with Alzheimer's LRP6 phosphorylation. Science 2008;321:1350–3. disease. Dementia 1994;5:6–11. 21.QinYB,LiL,PanWJ,WuDQ.Regulation of phosphatidylinositol 7. Zubenko GS, Stiffler JS, Hughes HB, Martinez AJ. Reductions in brain kinases and metabolism by Wnt3a and Dvl. J Biol Chem 2009; phosphatidylinositol kinase activities in Alzheimer's disease. Biol Psych 284:22544–8. 1999;45:731–6. 22. Wu P, Hu Y. Small molecules targeting phosphoinositide 3-kinases. 8. Landman N, Jeong SY, Shin SY, Voronov SV, Serban G, Kang MS, et al. MedChemComm 2012;3:1337–55. Presenilin mutations linked to familial Alzheimer's disease cause an 23. Zheng Z, Amran SI, Thompson PE, Jennings IG. Isoform-selective inhibi- imbalance in phosphatidylinositol 4,5-bisphosphate metabolism. Proc tion of phosphoinositide 3-kinase: identification of a new region of Natl Acad Sci U S A 2006;103:19524–9. nonconserved amino acids critical for p110alpha inhibition. Mol Phar- 9. Olsen HL, Hoy M, Zhang W, Bertorello AM, Bokvist K, Capito K, et al. macol 2011;80:657–64. Phosphatidylinositol 4-kinase serves as a metabolic sensor and regulates 24. Knight ZA, Gonzalez B, Feldman ME, Zunder ER, Goldenberg DD, Wil- priming of secretory granules in pancreatic beta cells. Proc Natl Acad Sci U S liams O, et al. A pharmacological map of the PI3-K family defines a role for A 2003;100:5187–92. p110a in insulin signaling. Cell 2006;125:733–47. 10. Minogue S, Waugh MG. The phosphatidylinositol 4-kinases: don't call it a 25. Clarke JH, Giudici ML, Burke JE, Williams RL, Maloney DJ, Marugan J, et al. comeback. Subcell Biochem 2012;58:1–24. The function of phosphatidylinositol 5-phosphate 4-kinase gamma 11. Wang J, Sun HQ, Macia E, Kirchhausen T, Watson H, Bonifacino JS, et al. (PI5P4Kgamma) explored using a specific inhibitor that targets the PI4P promotes the recruitment of the GGA adaptor proteins to the trans- PI5P-binding site. Biochem J 2015;466:359–67. Golgi network and regulates their recognition of the ubiquitin sorting 26. Zhou Q, Li J, Yu H, Zhai Y, Gao Z, Liu Y, et al. Molecular insights into the signal. Mol Biol Cell 2007;18:2646–55. membrane-associated phosphatidylinositol 4-kinase IIalpha. Nat Com- 12. Jovic M, Kean MJ, Dubankova A, Boura E, Gingras AC, Brill JA, et al. mun 2014;5:3552. Endosomal sorting of VAMP3 is regulated by PI4K2A. J Cell Sci 2014; 27. Tai AW, Bojjireddy N, Balla T. A homogeneous and nonisotopic assay for 127:3745–56. phosphatidylinositol 4-kinases. Anal Biochem 2011;417:97–102. 13. Henmi Y, Morikawa Y, Oe N, Ikeda N, Fujita A, Takei K, et al. PtdIns4- 28. Lu Y, Nikolovska-Coleska Z, Fang X, Gao W, Shangary S, Qiu S, et al. KIIalpha generates endosomal PtdIns(4)P and is required for receptor Discovery of a nanomolar inhibitor of the human murine double minute 2 sorting at early endosomes. Mol Biol Cell 2016;27:990–1001. (MDM2)-p53 interaction through an integrated, virtual database screening 14. Pizarro-Cerda J, Payrastre B, Wang YJ, Veiga E, Yin HL, Cossart P. Type II strategy. J Med Chem 2006;49:3759–62. phosphatidylinositol 4-kinases promote Listeria monocytogenes entry 29. Lu W, Liu X, Cao X, Xue M, Liu K, Zhao Z, et al. SHAFTS: a hybrid approach into target cells. Cell Microb 2007;9:2381–90. for 3D molecular similarity calculation. 2. Prospective case study in the 15. Simons JP, Al-Shawi R, Minogue S, Waugh MG, Wiedemann C, Evangelou discovery of diverse p90 ribosomal S6 protein kinase 2 inhibitors to S, et al. Loss of phosphatidylinositol 4-kinase 2alpha activity causes late suppress cell migration. J Med Chem 2011;54:3564–74.

www.aacrjournals.org Cancer Res; 77(22) November 15, 2017 6265

Li et al.

30. Jones G, Willett P, Glen RC. Molecular recognition of receptor sites using a 45. Sinha RK, Patel RY, Bojjireddy N, Datta A, Subrahmanyam G. Epigalloca- genetic algorithm with a description of desolvation. J Mol Biol 1995;245: techin gallate (EGCG) inhibits type II phosphatidylinositol 4-kinases: a key 43–53. component in pathways of phosphoinositide turnover. Arch Biochem 31. Jones G, Willett P, Glen RC, Leach AR, Taylor R. Development and Biophys 2011;516:45–51. validation of a genetic algorithm for flexible docking. J Mol Biol 46. Srivastava R, Ratheesh A, Gude RK, Rao KV, Panda D, Subrahmanyam G. 1997;267:727–48. Resveratrol inhibits type II phosphatidylinositol 4-kinase: a key compo- 32. Verdonk ML, Cole JC, Hartshorn MJ, Murray CW, Taylor RD. Improved nent in pathways of phosphoinositide turn over. Biochem Pharmacol protein–ligand docking using GOLD. Proteins 2003;52:609–23. 2005;70:1048–55. 33. Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, et al. 47. Balla A, Balla T. Phosphatidylinositol 4-kinases: old enzymes with emerg- Automated docking using a Lamarckian genetic algorithm and an ing functions. Trends Cell Biol 2006;16:351–61. empirical binding free energy function. J Comput Chem 1998;19: 48. Minogue S, Anderson JS, Waugh MG, dos Santos M, Corless S, Cramer R, 1639–62. et al. Cloning of a human type II phosphatidylinositol 4-kinase reveals a 34. Huey R, Morris GM, Olson AJ, Goodsell DS. A semiempirical free energy novel lipid kinase family. J Biol Chem 2001;276:16635–40. force field with charge-based desolvation. J Comput Chem 2007;28: 49. Waugh MG, Minogue S, Blumenkrantz D, Anderson JS, Hsuan JJ. Identi- 1145–52. fication and characterization of differentially active pools of type IIalpha 35. Martinez Molina D, Jafari R, Ignatushchenko M, Seki T, Larsson EA, Dan C, phosphatidylinositol 4-kinase activity in unstimulated A431 cells. Bio- et al. Monitoring drug target engagement in cells and tissues using the chem J 2003;376:497–503. cellular thermal shift assay. Science 2013;341:84–7. 50. Sellappan S, Grijalva R, Zhou X, Yang W, Eli MB, Mills GB, et al. Lineage 36. Jafari R, Almqvist H, Axelsson H, Ignatushchenko M, Lundback T, Nor- infidelity of MDA-MB-435 cells: expression of melanocyte proteins in a dlund P, et al. The cellular thermal shift assay for evaluating drug target breast cancer cell line. Cancer Res 2004;64:3479–85. interactions in cells. Nat Protoc 2014;9:2100–22. 51. Ellison G, Klinowska T, Westwood RF, Docter E, French T, Fox JC. Further 37. Choi S, Hedman AC, Sayedyahossein S, Thapa N, Sacks DB, Anderson RA. evidence to support the melanocytic origin of MDA-MB-435. Mol Pathol Agonist-stimulated phosphatidylinositol-3,4,5-trisphosphate generation 2002;55:294–9. by scaffolded phosphoinositide kinases. Nat Cell Biol 2016;18:1324–35. 52. Ross DT, Scherf U, Eisen MB, Perou CM, Rees C, Spellman P, et al. 38. Sun F, Li J, Chen C. Puzzle out the regulation mechanism of PI4KIIalpha Systematic variation in gene expression patterns in human cancer cell activity. Sci China Life Sci 2014;57:636–8. lines. Nat Genet 2000;24:227–35. 39. Miduturu CV, Deng X, Kwiatkowski N, Yang W, Brault L, Filippako- 53. Zeitouni D, Pylayeva-Gupta Y, Der CJ, Bryant KL. KRAS mutant pancreatic poulos P, et al. High-throughput kinase profiling: a more efficient cancer: no lone path to an effective treatment. Cancers 2016;8. doi: approach toward the discovery of new kinase inhibitors. Chem Biol 10.3390/cancers8040045. 2011;18:868–79. 54. Downward J.Targeting RAS and PI3K in lung cancer. Nat Med 2008; 40. Hollestelle A, Elstrodt F, Nagel JH, Kallemeijn WW, Schutte M. Phospha- 14:1315–6. tidylinositol-3-OH kinase or RAS pathway mutations in human breast 55. Engelman JA, Chen L, Tan X, Crosby K, Guimaraes AR, Upadhyay R, et al. cancer cell lines. Mol Cancer Res 2007;5:195–201. Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and 41. Torbett NE, Luna-Moran A, Knight ZA, Houk A, Moasser M, Weiss W, et al. A PIK3CA H1047R murine lung cancers. Nat Med 2008;14:1351–6. chemical screen in diverse breast cancer cell lines reveals genetic enhancers 56. Junttila MR, Devasthali V, Cheng JH, Castillo J, Metcalfe C, Clermont AC, and suppressors of sensitivity to PI3K isoform-selective inhibition. Bio- et al. Modeling targeted inhibition of MEK and PI3 kinase in human chem J 2008;415:97–110. pancreatic cancer. Mol Cancer Ther 2015;14:40–7. 42. Graham TR, Burd CG. Coordination of Golgi functions by phosphatidy- 57. Toulany M, Iida M, Keinath S, Iyi FF, Mueck K, Fehrenbacher B, et al. Dual linositol 4-kinases. Trends Cell Biol 2011;21:113–21. targeting of PI3K and MEK enhances the radiation response of K-RAS 43. Wang YJ, Wang J, Sun HQ, Martinez M, Sun YX, Macia E, et al. Phospha- mutated non-small cell lung cancer. Oncotarget 2016;7:43746–61. tidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 58. Minogue S, Waugh MG, De Matteis MA, Stephens DJ, Berditchevski F, complexes to the Golgi. Cell 2003;114:299–310. Hsuan JJ. Phosphatidylinositol 4-kinase is required for endosomal traf- 44. Pitts TM, Newton TP, Bradshaw-Pierce EL, Addison R, Arcaroli JJ, Klauck PJ, ficking and degradation of the EGF receptor. J Cell Sci 2006;119:571–81. et al. Dual pharmacological targeting of the MAP kinase and PI3K/mTOR 59. Ketel K, Krauss M, Nicot AS, Puchkov D, Wieffer M, Muller R, et al. A pathway in preclinical models of colorectal cancer. PLoS One 2014;9: phosphoinositide conversion mechanism for exit from endosomes. Nature e113037. 2016;529:408–12.

6266 Cancer Res; 77(22) November 15, 2017 Cancer Research

PI-273, a Substrate-Competitive, Specific Small-Molecule Inhibitor of PI4KII α, Inhibits the Growth of Breast Cancer Cells

Jiangmei Li, Zhen Gao, Dan Zhao, et al.

Cancer Res 2017;77:6253-6266. Published OnlineFirst August 21, 2017.

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