Therapeutics, Targets, and Chemical Biology

PI-273, a Substrate-Competitive, Specific Small-Molecule Inhibitor of PI4KIIa, Inhibits the Growthof Breast Cancer CellsJiangmei Li1,2, Zhen Gao1,3, 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) has beenidentified as a potential target for antitumor therapy, the clinicalapplications of PI4KIIa are limited by a lack of specific inhibi-tors. Here we report the first small-molecule inhibitor(SMI) of human PI4KIIa. Docking-based and ligand-basedvirtual screening strategies were first employed to identifypromising hits, followed by two rounds of kinase activityinhibition validation. 2-(3-(4-Chlorobenzoyl)thioureido)-4-ethyl-5-methylthiophene-3-carboxamide (PI-273) exhibitedthe greatest inhibitory effect on PI4KIIa kinase activity(IC50 ¼ 0.47 mmol/L) and suppressed cell proliferation. Surfaceplasmon resonance and thermal shift assays indicated that PI-273 interacted directly with PI4KIIa. Kinetic analysis identifiedPI-273 as a reversible competitive inhibitor with respect to the

substrate phosphatidylinositol (PI), which contrasted withmost other PI kinase inhibitors that bind the ATP binding site.PI-273 reduced PI4P content, cell viability, and AKT signaling inwild-typeMCF-7 cells, but not in PI4KIIa knockoutMCF-7 cells,indicating that PI-273 is highly selective for PI4KIIa. Mutantanalysis revealed a role of palmitoylation insertion in theselectivity of PI-273 for PI4KIIa. In addition, PI-273 treatmentretarded cell proliferation by blocking cells in G2–M, inducingcell apoptosis and suppressing colony-forming ability. Impor-tantly, PI-273 significantly inhibited MCF-7 cell-induced breasttumor growth without toxicity. PI-273 is the first substrate-competitive, subtype-specific inhibitor of PI4KIIa, the use ofwhich will facilitate evaluations of PI4KIIa as a cancer thera-peutic target. Cancer Res; 77(22); 6253–66. �2017 AACR.

IntroductionPhosphatidylinositol (PI) is an essential phospholipid that

serves as a metabolic precursor for both phosphoinositides andinositol (Ins) phosphates (1), and its related phosphatidylino-sitol kinases (PIK) play important roles in a number of human

diseases, including cancer (2–4), malaria (5), Alzheimer disease(6–8), and diabetes (9). Among the phosphatidylinositolkinases, PI4KIIa is targeted to the trans-Golgi network (TGN)and endosomes. In mammalian cells, PI4KIIa is the dominantlipid kinase generating PI4-phosphate (PI4P; ref. 10), which isnot only the precursor of the important regulatory phosphoi-nositides PI(4,5)P2 and PI(3,4,5)P3 but also is an emergingregulatory molecule in trans-Golgi (11) and endosomal traf-ficking (12, 13), as well as phagocytosis (14). PI4KIIa knockoutmice develop late-onset neurologic features that resemblehuman hereditary spastic paraplegia (15). Recently, an increas-ing number of reports have identified PI4KIIa as a potentialtarget for breast cancer therapy (3, 4, 16–19). We previouslyreported that increased PI4KIIa expression promotes tumorgrowth by altering the HER2/PI3-kinase (PI3K)/ERK signalingcascades (4) and that dual inhibition of EGFR and PI4KIIarepresents a novel strategy to combat EGFR-dependent tumors(17). PI4KIIa is also important for the Wnt (20, 21) and Akt (3)signaling pathways. However, in contrast to the extensive studyon PI3Ks, pharmacologic manipulation and basic scientificresearch on PI4KIIa has been limited, largely owing to a lackof specific inhibitors.

Numerous small-molecule inhibitors (SMI) of PIKs havebeen identified via screening in the past two decades, but mostare ATP-competitive inhibitors of PI3Ks (22). Only 2-methyl-5-nitro-2-[(6-bromoimidazo[1,2-a]pyridin-3-yl)methylene]-1-methylhydrazide-benzenesulfonic acid (PIK-75; refs. 23, 24)and NCGC00012848-02 (NIH-12848; ref. 25) have beenidentified as substrate-competitive inhibitors. Isoform selec-tivity is complicated by high conservation of the nucleotide-

1National Laboratory of Biomacromolecules, CAS Center for Excellence inBiomacromolecules, Institute of Biophysics, Chinese Academy of Sciences,Chaoyang District, Beijing, China. 2Shanghai Institute for Advanced Immuno-chemical Studies, ShanghaiTech University, Shanghai, China. 3University ofChinese Academy of Sciences, Shijingshan District, Beijing, China. 4Drug Dis-covery and Design Center, State Key Laboratory of Drug Research, ShanghaiInstitute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.5Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences,Shanghai, China. 6The State Key Laboratory of Toxicology andMedical Counter-measures, Academy of Military Medical Science, Beijing, China. 7Beijing Institutefor Brain Disorders, You An Men, Beijing, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

J. Li, Z. Gao, and D. Zhao contributed equally to this article.

Corresponding Authors: Chang Chen, National Laboratory of Biomacromole-cules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road,Chaoyang District, Beijing 100101, P.R. China. Phone: 8610-6488-8406; Fax:8610-6487-1293; E-mail: changchen@moon.ibp.ac.cn; and Cheng Luo, DrugDiscovery and Design Center, State Key Laboratory of Drug Research, ShanghaiInstitute of Materia Medica, Chinese Academy of Sciences, 555 ZuchongzhiRoad, Pudong District, Shanghai, 201203, PR China. Phone: 86-21-5027-1399;E-mail: cluo@simm.ac.cn

doi: 10.1158/0008-5472.CAN-17-0484

�2017 American Association for Cancer Research.

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binding pocket amino acid sequences of the differentPIK isoforms, the site at which most SMIs bind. Becauseisoform-selective inhibitors may reduce toxicity by decreas-ing off-target effects, substrate-competitive inhibitors areurgently needed.

By combining crystal structure data, molecular dynamics (MD)simulations, andbiochemical andmutagenesis studies, we recent-ly identified the putative PI-binding pocket for PI4KIIa (26). ThePI-binding pocket is lined by residues E157, Y159, R181, L184,T307, D308, and L349 on the palmitoylation insertion andactivation loop. In addition to contributing to the PI-bindingpocket, the palmitoylation insertion also interacts with the mem-brane via an amphipathic helix, a4, and any perturbation thatweakens its membrane interaction significantly impairs its kinaseactivity. This insertion does not occur in other PIKs, making it anideal site for screening specific inhibitors. In addition, a high-throughput andnonisotopic assay for PI4-kinase activity has beendeveloped (27), thereby enabling screens for PI4KIIa-specificinhibitors among large compound libraries. Structure-based vir-tual screening (SBVS) has become apowerful tool in the tool kit ofmedicinal chemists for rapidly enriching hits from large pools ofvirtual compound libraries (28). In this study, we identified a PIsubstrate-competitive SMI for PI4KIIa that notably inhibits tumorgrowth in a breast cancer model without toxicity.

Materials and MethodsVirtual screening

The SPECS database (http://www.specs.net) is a compoundlibrary of 199,970 single-synthesized, well-characterized smallmolecules. This database has been widely used in the study ofcompound screening processes and drug discovery and wasused as the ligand database in this study. To refine the databaseto include compounds with good drug-like properties [(molec-ular weight� 500, log P � 5, number of hydrogen bond donors� 7, number of hydrogen bond acceptors � 14), molecularsolubility � –7 (expressed as log S, where S is the solubility inmol/L), ADMET solubility � –5 (base 10 logarithm of molarsolubility as predicted by regression), ADMET solubility level �2 (assigns the molecule to one of seven solubility classes basedon the ADMET solubility value)], we filtered the database usingPipeline Pilot 7.5 (Scitegic, Inc.) prior to screening. The filtereddatabase was used for a subsequent SHAFTS-3D ligand simi-larity search. The SHAFTS algorithm was implemented in theChemMapper web server, which is a hybrid 3D molecularsimilarity calculation approach designed to combine thestrength of pharmacophore matching and volumetric overlaythat exhibits satisfactory "scaffold hopping" capability againstseveral representative kinases and has been utilized for ligand-based virtual screening (29). As PI stearoyl-arachidonyl acylchains are long and flexible, a substituted compound with abutyryl group attached to the oxygen atom of both PI sidechains was used. The structure of the substituted compoundwas drawn with ChemBio Office 2015, and MM2minimizationwas used to obtain the starting structure for the similaritysearch. Using the substituted compound as the SHAFTS inputmolecule, the top 1,000 molecules with similarity scores > 1.0were reserved, resulting in 822 candidates. The crystal structureof human PI4KIIa (PDB entry: 4HNE; ref. 26) was used as thereceptor for molecular docking. The docking accuracies of theGLIDE 5.5 (Glide, version 5.5, Schr€odinger, LLC), Gold 5.0

(30–32) and Autodock 4.2 (33, 34) programs were evaluated.The substituent PI molecule was mixed with 50 decoy mole-cules and docked into the PI-binding site using the threesoftware programs. Because the GLIDE 5.5 software providedthe best PI substituent ranking, this program was used forsubsequent structural-based virtual screening. Docking studiesin Gold 5.0 were performed using the default genetic algorithm(GA) parameters and automatic settings with 100% searchefficiency. The kinase scoring function implemented in Goldwas applied in the docking calculations, and GA docking wasset to be terminated if the best three solutions were all within1.5 Å root mean square deviations (RMSD) of each other. InAutodock 4.2, the 50 decoy molecules and the PI substituentwere docked to PI4KIIa using the Lamarckian GA, and a 0.375Å grid spacing was set for the energetic map calculations. Thenumber of GA runs was set to 10, and the standard precision(SP) GLIDE 5.5 mode was used. The protein structure wasprepared using the Protein Preparation Wizard Workflow withdefault settings. The PAL loop and activation loop were definedas the PI-binding site in which the docking grids were created(26). The 822 compounds were docked into the defined bind-ing site and ranked on the basis of their GLIDE score (G-score).Considering both the docking scores and binding modes, 522compounds were selected for further evaluation, which werethen structurally clustered into 60 clusters based on their 2Dmolecular fingerprints using the Cluster Molecules module inPipeline Pilot 7.5. Two or three candidate molecules withrelatively simple chemical structures and higher G-scores with-in each structural cluster were retained. Ultimately, a total of142 compounds were selected and purchased for the first-roundbiochemical assay. On the basis of the structures of the 7 activecompounds identified by the first-round biochemical assay, theSHAFTS method was again utilized for scaffold hopping (29). Atotal of 53 candidates were purchased for the second-roundbiochemical assay.

Expression and purification for PI4KIIa, PI4KIIb, and PI4KIIIbThe constructs PI4KIIa, PI4KIIb, and PI4KIIIb were expressed

as GST fusion proteins in Escherichia coli BL21-CodonPlus(DE3) competent cells. The cells were grown at 37�C until anOD600 of 1.0 was reached, at which point they were induced by0.3 mmol/L isopropyl b-D-thiogalactoside at 16�C for 18 hours.The cells were then harvested and resuspended in buffer con-taining 50 mmol/L HEPES (pH 7.5), 1 mol/L NaCl, 2 mmol/LDTT, 20 mmol/L MgCl2, 1 mmol/L phenylmethylsulfonylfluoride, and 1 mg/mL lysozyme. Cell suspensions werehomogenized with a high-pressure cell disruptor (JN BIO) at30,000 psi, and cell debris was removed by centrifugation at120,000� g for 40 minutes. Proteins were purified using a GST-affinity column (GE Healthcare), and the GST tag was cleavedovernight at 4�C with PreScission protease (GE Healthcare) inbuffer containing 50 mmol/L HEPES (pH 7.5), 300 mmol/LNaCl, 0.1% Triton X-100, and 2 mmol/L MgCl2.

Kinases activity assayThe proteins used in this study were purchased from the

following vendors: PI4KIIIa from Creative BioMart (catalog no.PI4KA-161H), PI3Ka from Invitrogen (catalog no. PV4788),PIK3Cd from Millipore (catalog no. 14-604-K), PIK3Cb fromMillipore (catalog no. 14-603-K), PIK3Cg from Invitrogen (cat-alog no. PR8641C), AKT1 from BPS (catalog no. 40003), AKT2

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from Carna (catalog no. 01-102), and AKT3 from Carna (catalogno. 01-103, Lot. No. 05CBS-3274). In addition, PI4KIIa, PI4KIIb,and PI4KIIIbwere expressed and purified as described above. TheADP-Glo kinase assay (Promega) was used to evaluate the kinaseactivities of PI4KIIa, PI4KIIb, PI4KIIIa, PI4KIIIb, PI3Ka, PI3Kd,PI3Kb, and PI3Kg according to previous reports with some mod-ifications (27). Briefly, 5 mL of kinase buffer containing differentconcentrations of compounds (25 nmol/L PI4KIIa, 268 nmol/LPI4KIIb, 83 nmol/L PI4KIIIa, 109 nmol/L PI4KIIIb, 1.65 nmol/LPI3Ka, 4.8nmol/L PI3Kb, 7.6nmol/L PI3Kg , or 17nmol/L PI3Kd)was added to each well of the assay plate. For the PI4K assay, thebuffer contained 50mmol/LHEPES (pH 7.5), 150mmol/L NaCl,0.1% anzergent 3–14, 1 mmol/L EDTA, and 20 mmol/L MgCl2,while the buffer for the PI3K assay contained 50 mmol/L HEPES(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 wasinitiated by adding 5 mL of the substrate solution (50 mmol/LPI(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 thencovered and incubated at room temperature for 1 hour, and thereactions were stopped by adding 10 mL of ADP-Glo reagent(Promega). After equilibration for 40 minutes, 20 mL of kinasedetection reagent (Promega) was added, and the mixture (40 mLtotal) was incubated for an additional 60 minutes before readingthe luminescence on a Varioskan Flash plate reader (ThermoScientific).

The kinase activities of AKT1, AKT2, and AKT3 were mea-sured by a mobility shift assay. Briefly, 15 mL of kinase buffer[50 mmol/L HEPES (pH 7.5), 0.0015% Brij-35] containingdifferent concentrations of the compounds (2.5 nmol/L AKT1,0.5 nmol/L AKT2 or 0.7 nmol/L AKT3) was added to each wellof the assay plate. Then, a mixture consisting of 10 mL of FAM-labeled peptide (GL Biochem, 3 mmol/L) and ATP (60 mmol/L,188 mmol/L, or 67 mmol/L) was added to the assay plate, whichwas then incubated at room temperature for 30 minutes.Finally, the reaction was stopped by adding 25 mL of stopbuffer [100 mmol/L HEPES (pH 7.5), 0.015% Brij-35, 0.2%coating reagent, 50 mmol/L EDTA], and the data were collectedwith the Caliper instrument (Perkin Elmer).

Thermal shift assay and cellular thermal shift assayThe thermal shift assay was performed on a 7500 Fast Real-

Time PCR system (Applied Biosystems). Each reaction solutioncontained 5 mmol/L PI4KIIa, 5� SYPRO orange (Sigma-Aldrich)and the test compounds in 20 mL of buffer [50 mmol/L HEPES(pH7.5), 150mmol/LNaCl, 2mmol/LMgCl2], whichwas heatedfrom 25�C to 95�C at a 1% ramp rate. The melting temperature(Tm) was calculated by the Boltzmann fitting method usingProtein Thermal Shift Software Version 1.1 (Applied Biosystems).Each reaction was repeated three times.

The cellular thermal shift assay (CETSA) was carried outbased on a previously described protocol with minor modifica-tions (35, 36). For the in vivo assay, MCF-7 cells were harvestedand washed with PBS after the indicated treatments. All bufferswere supplemented with complete protease inhibitor cocktail,and the cell suspensions were freeze-thawed three times usingliquid nitrogen. The soluble fraction (lysate) was separatedfrom the cell debris by centrifugation at 20,000 � g for 30minutes at 4�C. For the in vitro assay, the cell lysates weredivided into two aliquots; one aliquot was treated with PI-273,and the other was treated with the diluent DMSO (control) for

30 minutes. The lysates were then divided into smaller (50 mL)aliquots and heated individually at different temperatures for3 minutes (S1000TM thermal cycler, Bio-Rad), followed bycooling for 3 minutes at room temperature. The appropriatetemperatures were determined in preliminary CETSA experi-ments. The heated lysates were centrifuged at 20,000 � g for 30minutes at 4�C to separate the soluble fractions from theprecipitates. The supernatants were transferred to new micro-tubes and analyzed by SDS-PAGE followed by Western blotanalysis.

Detection and quantification of cellular PI(4)P, LPA,PI3P, PI(4,5)P2, PI(3,4,5)P3

All the cellular lipid contents were measured using the appro-priate Mass ELISA kits (Echelon Biosciences Inc.) according to themanufacturer's instructions. The kits used included the PI(4)PMass ELISA Kit (catalog no. K4000E), the LPA Mass ELISA Kit(catalog no. K-2800S), the PI3P Mass ELISA Kit (catalog no. K-3300), the PI(4,5)P2Mass ELISA Kit (catalog no. K-4500), and thePI(3,4,5)P3Mass ELISA Kit (catalog no. K-2500S). Briefly, cellularlipid extracts were analyzed for PI(4)P, LPA, PI3P, PI(4,5)P2 or PI(3,4,5)P3 using the appropriate Mass ELISA Kit described aboveand normalized by the protein concentration in the cell lysate. Formeasurement of PI4P in tumor tissues, lipids extracted fromequalweights (approximately 10 mg) of xenograft tumors were ana-lyzed for PI4P using the PI4PMass ELISA Kit described above andnormalized by the tissue weights. Statistical analysis was per-formed with 6 paired cases (37).

Surface plasmon resonance–based binding assayThe surface plasmon resonance (SPR) binding assays were

performed on a Biacore T200 instrument (GE Healthcare) at25�C. The PI4KIIa protein was covalently immobilized on a CM5chip using a standard amine-coupling procedure in 10 mmol/Lsodiumacetate (pH5.0). The chipwasfirst equilibratedwithHBS-EP buffer [10 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 3mmol/L EDTA, 0.05% (v/v) surfactant P20, 0.1%(v/v) DMSO]overnight. The compounds were serially diluted in HBS-EP bufferand injected for 120 seconds (contact phase), followed by 120seconds for the dissociation phase. The Kd values of the testedcompounds were determined using the Biacore T200 evaluationsoftware (GE Healthcare).

Mouse xenograft experimentsEight-week-old male BALB/c nude mice (Weitonglihua) were

allowed to acclimate for 1 week under specific pathogen-freeconditions in the animal facility of the Institute of Biophysics atthe Chinese Academy of Sciences (Beijing, China). The mice weresubcutaneously injected with an MCF-7 cell suspension in amixture of Matrigel and DMEM, and treatment was initiated after2 or 4 days. PI-273 was reconstituted in DMSO and administeredby intraperitoneal injection. Tumor volume was monitored viadigital calipers three times each week and calculated using theformula (smallest diameter2� largest diameter)/2. All proceduresinvolving animals and their care were approved by the animalethics committee of the Institute of Biophysics at the ChineseAcademy of Sciences (Beijing, China).

PharmacokineticsPI-273 was formulated in a 0.9% saline solution containing

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 formulatedPI-273, and another three were intragastrically treated with10 mL/kg formulated PI-273. Blood samples were collected at0.08, 0.16, 0.33, 0.67, 1, 1.5, 2, 3, and 5 hours after the intrave-nous and intragastrical administrations. One 0.2-mL blood sam-ple was collected from each rat by retro-orbital bleeding. Bloodwas collected into heparin-containing tubes, and plasma wasobtained by centrifugation at 4,000 rpm for 10 minutes. Allprotocols were based on standard operating procedures approvedby the Institutional Animal Care and Use Committee (IACUC)of the Shanghai Institute of Materia Medica at the ChineseAcademy of Sciences (Beijing, China). The plasma concentrationsof PI-273 were quantitated by the LC/MS-MS method, and non-compartmental analysis using Phoenix 1.3 (Pharsight) was per-formed for all analytic measurements. The trapezoidal methodwas used to calculate the area under the concentration–time curve(AUC), where AUC0–¥ ¼ AUC0-¼t þ Ct/ke (ke is the eliminationrate constant). The mean residence time [(MRT) ¼ AUMC/AUC]and the elimination half-life [(t1/2) ¼ 0.693/ke] were alsocalculated.

Statistical analysisStatistical analyses were performed using two-tailed paired

Student t test. Differences were considered statistically significantwhen the P value was <0.01, as indicated in the legends. All dataare expressed as the means � SD.

ResultsIdentification of PI-273 as a PI4KIIa inhibitor based on virtualscreening

In this study, virtual screening was employed to identify prom-ising hits (Fig. 1A). The SPECS database (https://www.specs.net),containing 199,970 compounds, was used as the ligand database,and compounds with unfavorable physicochemical propertieswere filtered using Pipeline Pilot 7.5. The remaining 89,467compounds were used in a SHAFTS-3D ligand similarity searchwith a PI-substituted compound as the input molecule, resultingin 822 candidates. The 822 compounds were docked into thePI4KIIa protein pocket near the palmitoylation insertion usingtheGLIDE 5.5 program.On the basis of the G-score and structuralclustering, 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 structuresof 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 indicatedtimes. 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 collectedfor the indicated lipids analyses. The values are presented as the means � SD from three independent experiments, and all the above experiments wereperformed three times in triplicate. � , P < 0.01.

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purchased for biochemical assays. We then tested the effects ofthese compounds on PI4KIIa activity at 100 mmol/L and identi-fied 7 that exhibited greater than 60% kinase activity inhibition(Supplementary Fig. S1A). Measuring the half-maximal inhibi-tory concentration (IC50) values of these compounds for kinaseactivity in vitro (Supplementary Fig. S1B) and cell viability in vivo(Supplementary Fig. S1C) revealed that PI-93 (SupplementaryFig. S1D) exhibited the strongest inhibitory effect.We then furthertested the effect of PI-93 on the intracellular lipid content. Asshown in Supplementary Fig. S1E, theMCF-7 cells incubatedwith20 mmol/L PI-93 for 24 hours exhibited lower PI4P levels thanDMSO-treated cells, while the LPA, PI3P, PI(4,5)P2, and PI(3,4,5)P3 levels were not significantly different in either sample.

On the basis of the SHAFTS-3D ligand similarity search, anoth-er 53 candidates were selected and purchased for the secondround of screening. Six compounds with an inhibitory effect onPI4KIIa activity stronger than that of PI-93 were selected forfurther analysis (Supplementary Fig. S2A; Fig. 1A). The chemicalstructures of these representative PI-93 analogues are presented inSupplementary Table S1 (in addition to the 6 selected com-pounds, 5 compounds that shared a common parent nucleuswith PI-93 in the first-round screening, and another 3 compoundsthat had an inhibition effect similar to that of PI-93 in the second-round screening have also been included) and Fig. 1B. Thesuperimposition of PI with these six representative active com-pounds is shown in Supplementary Fig. S2B. The IC50 values ofthe compounds of interest for PI4KIIa kinase activity in vitroand cell viability in vivo in MCF-7 cells are presented in Supple-mentary Table S1. 2-(3-(4-chlorobenzoyl)thioureido)-4-ethyl-5-methylthiophene-3-carboxamide (PI-273) had the greatest inhib-itory effect of MCF-7 cell viability, and its IC50 value in thebiochemical assay was 0.47 mmol/L. In addition, our results(Supplementary Fig. S2C) also indicated that PI-273 had the bestinhibitory effect on the cellular PI4P content among the analogues(PI-294, PI295, PI-308), which was consistent with the analogueseffects on cell viability and AKT activity. Other PI-273 analogues,such as PI-69/107/114, that had no effect on PI4KIIa activity alsohad no effect on MCF-7 cells (Supplementary Table S1). In thePI4P content assay, PI-273 reduced the intracellular PI4P contentin MCF-7 cells in a dose- and time-dependent manner (Fig. 1C).Furthermore, as shown in Fig. 1D, PI-273 treatment could spe-cifically inhibit PI4P production but not that of other lipids, suchas LPA, PI3P, PI(4,5)P2 and PI(3,4,5)P3. Taken together, theseresults suggest that PI-273 is a structurally novel PI4KIIa inhibitorwith remarkable potency against the kinase activity of PI4KIIa.

PI-273 directly binds and reversibly inhibits PI4KIIaTo validate these potential PI4KIIa inhibitors, direct interac-

tions between PI4KIIa and the candidate compounds were mea-suredby SPR.As shown inFig. 2AandSupplementary Fig. S3A, theinteractions were dose-dependent. The equilibrium dissociationconstant (Kd) between PI-273 and PI4KIIa was approximately9.49 mmol/L based on the association rate constant (kon¼ 2.08 �102 M�1s�1) and the dissociation rate constant (koff ¼ 2.13 �10�3 s�1), whereas theKd of PI-277was 9.59mmol/L and theKd ofPI-282 was 5.91 mmol/L (these two compounds have the bestsolubility among PI-273/274/275/276/282/294/295/308; listedin Supplementary Table S1). The direct interaction between thesecompounds and PI4KIIawas further confirmed by a fluorescence-based thermal shift assay; PI-273 dose dependently shifted themelting temperature (Tm) of PI4KIIa by more than 4�C before

reaching a plateau. This effect was also observed in samplestreated with PI-93, PI-277, and PI-294, whereas PI-69 did notaffect the PI4KIIa Tmvalue (Fig. 2B; Supplementary Fig. S3B),consistent with their individual potencies for PI4KIIa inhibition.To evaluate the in vivo interaction between PI-273 andPI4KIIa, weconducted a CETSA (35). The CETSA, developed by D. MolinaMartinez and his colleagues, is used for assessing target engage-ment by drugs in vivo. The CETSA is based on the biophysicalprinciple of the ligand-induced thermal stabilization of targetproteins, and it will likely become a valuable tool for validatingand optimizing drug target engagement (35, 36). As shown in Fig.2C, intact MCF-7 cells treated with 1 mmol/L PI-273 for 96 hoursand cell lysate treated with 20 mmol/L PI-273 for 30 minutesexhibited shifted melting curves for PI4KIIa. Under DMSO treat-ment, 50% of PI4KIIa was degraded at 60�C, whereas in the PI-273–treated samples, 50% degradation required temperatures of70�C (intact cells) and 75�C (cell lysate). Thus, PI-273 inhibitsPI4KIIa activity via a direct binding event. We then assessed thereversibility of the binding between PI-273 and PI4KIIa. PI4KIIawas preincubated with varying concentrations of PI-273 (0–1mmol/L) for different lengths of time (0–60 minutes) before thekinase reaction was initiated. Only the concentration of PI-273and not the incubation time influenced the inhibitory effect of PI-273 on PI4KIIa activity (Fig. 2D), suggesting that the interactionbetweenPI-273 andPI4KIIa is reversible. As further confirmation,we evaluated the enzyme kinetics of PI4KIIa, which revealed thatthe slope of the velocity-to-enzyme concentration decreased withincreasing PI-273 concentration (Fig. 2E), thus indicating that PI-273 is a reversible inhibitor of PI4KIIa. Taken together, theseresults strongly indicate that PI-273 directly interacts with PI4KIIain a reversible manner.

PI-273 is a PI-competitive inhibitor of PI4KIIaThere are three types of reversible inhibitors: competitive,

uncompetitive, and anticompetitive. As described above, PI-273 is a reversible inhibitor of PI4KIIa, and enzyme kineticanalysis was performed to determine the exact type of thisinhibition (Fig. 3A and B). At varying concentrations of ATP,increasing PI-273 decreased the maximum velocity (Vmax) of thereaction (Fig. 3A andC), but the apparent affinity (KM,Michaelis–Menten constant) of ATP for the binding site did not change (Fig.3A and D), indicating that PI-273 is an ATP-uncompetitiveinhibitor. However, when we varied the concentration of PI, theVmax of the reaction remainedunchanged (Fig. 3B andC),whereasthe KM value for PI at the binding site increased, indicating that PI-273 is a PI-competitive inhibitor (Fig. 3B and D). We nextperformed experiments with PI4KIIa mutants to verify this con-clusion (Fig. 3E; Supplementary Table S2). As expected, the Tmof wild-type PI4KIIa was significantly elevated in the presence ofPI-273. However, mutations or deletions in the PI-binding reg-ulation region (for example, the palmitoylation insertion, D165-173, or K165E/K168E/K172E), but not those at the nucleotide-binding site (for example, K152A, D346A), reduced the DTmvalue. We previously reported that the palmitoylation insertionmodulates kinase activity by tuning the PI-binding pocket (26,38), and these results confirm that PI-273 is a PI-competitiveinhibitor of PI4KIIa.

PI4KIIa is required for the inhibitory effect of PI-273The selectivity of kinase inhibitors is crucial to avoid side effects

caused by off-target inhibition (39). To validate the subtype-

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specific inhibitory effect of PI-273 on PI4KIIa, we tested theinhibitory effects of PI-273 on 11 related kinases, including fourPI4K subtypes (PI4KIIa, PI4KIIb, PI4KIIIa, and PI4KIIIb), fourPI3K subtypes (PI3Ka, PI3Kb, PI3Kg , PI3Kd) and three AKTsubtypes (AKT1, AKT2, and AKT3). As shown in Fig. 4A, the IC50

values of PI-273 for PI4KIIa were 0.47 mmol/L, consistent with

our previous results. The PI-273 IC50 values were much higher forthe other kinases than for PI4KIIa, even for the isoformhaving thegreatest homology to PI4KIIa, PI4KIIb, which had a PI-273 IC50

value more than 30-fold higher than that for PI4KIIa. For theother PI4K subtypes (PI4KIIIa and PI4KIIIb), whose structures aremore similar to those of PI3Ks, the IC50 values were both more

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/Lto 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 varyingconcentrations 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) andcell 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 ATPwas added. The graphs represent normalized percentage of inhibition compared with preincubation time. E, The initial velocity kinetics compared with varyingPI4KIIa 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 withcomparable results.

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than 100 mmol/L. Although the PI-273 IC50 value for PI3Kb wasonly 10-foldhigher than that for PI4KIIa, our results shown in Fig.1D indicate that PI-273 treatment cannot reduce PI3P or PI(3,4,5)P3 content, suggesting that PI3Kb is not amajor target of PI-273 invivo. All the above results indicate that PI-273 is highly specific forPI4KIIa.

We next tested the specificity of PI-273 for PI4KIIa in vivo. Wecarried out genome-scale CRISPR-Cas9 knockout screening for PI-273-resistant genes (Supplementary Fig. S4A). SupplementaryFigure S4B shows that PI4KIIa is the most significantly enrichedtarget, whose function loss can confer strong protection toMCF-7cells against PI-273. We then generated PI4KIIa knockout MCF-7cells using the CRISPR-Cas9 method (Supplementary Fig. S5). Asexpected, obtaining MCF-7 cells in which PI4KIIa was totallyknocked out proved to be difficult. Among 200 individual mono-clones, only one PI4KIIa knockout cell line was selected, and itssequencing results indicated that PI4KIIa exists as a multicopygene in MCF-7 cells (Supplementary Fig. S5A). We then furtherconfirmed the knockout effects of this clone by Western blotanalysis (Supplementary Fig. S5B) and immunofluorescence

staining (Supplementary Fig. S5C). In the cell viability assay, thePI4KIIa knockout greatly decreased the sensitivity to PI-273.Compared with the DMSO-treated wild-type MCF-7 cells, 10mmol/L PI-273 could reduce the cell viability by approximately80%, while this ratio for the PI4KIIa knockout MCF-7 cells wasonly 25% (Fig. 4B), indicating that PI4KIIa is essential for theeffect of PI-273 on cell viability. Because our previous works (4,17) and other studies indicated that PI4KIIa can regulate the AKTsignaling pathway (3), we tested the effects of our compounds onAKT signaling. As shown in Fig. 4C and Supplementary Fig. S6A,PI-273 could suppress the AKT signaling pathway in a dose- andtime-dependent manner. However, other analogues, such as PI-294 (Supplementary Fig. S6B), PI-250 (Supplementary Fig. S6C),PI-295, and PI-308 (Supplementary Fig. S6D) had much weakereffects on this signaling pathway, consistent with their effects oncell viability and PI4P content. These results indicate that PI4KIIais the target for PI-273. We next addressed whether PI4KIIa issolely essential for the effects of PI-273 in vivo using PI4KIIaknockout cells, and we found that PI-273 reduced the PI4Pcontent by 40% in wild-type (CTR) cells but had no effect on

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 varyingconcentrations of ATP (A) and PI (B). Double-reciprocal plots of initial velocities (Lineweaver–Burk plots) showing uncompetitive inhibition by PI-273 toward ATPand purely competitive inhibition toward PI as a substrate. The experiment was performed three times with comparable results. Vmax (C) and KM (D) values for ATPand 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 weretreated 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.

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PI4P in PI4KIIa knockout (KO) cells. In contrast, PI-273 had noeffect on other lipid [LPA, PI3P, PI(4,5)P2 or PI(3,4,5)P3] contentin wild-type cells or PI4KIIa knockout cells (Fig. 4D). Consistentwith this result, PI-273 decreased the p-AKT levels by 50% inwild-type cells but had no effect on their phosphorylated levels inPI4KIIa knockout cells (Fig. 4E). In addition, PI-273 did not affectthe PI4P level (Supplementary Fig. S7A) or the AKT signalingpathway (Supplementary Fig. S7B) in the PI4KIIa knockdowncells generated by siRNA. Taken together, the in vitro and in vivoexperimental results indicate that PI-273 is a PI4KIIa-specificinhibitor.

Effect of PI-273 on breast cancer cellsCompounds PI-273, PI-274, PI-277, PI-294, PI-295, and PI-

308 inhibited PI4KIIa activity both in vitro and in vivo and hadvarying effects on the viability of MCF-7 cells. PI-273 had thegreatest effect on PI4KIIa inhibition and was thus selectedfor further exploration. To examine the biological effects ofPI4KIIa inhibition in greater detail, we tested the inhibitory effectof PI-273 on different breast cancer cell lines: MCF-7, T-47D, SK-BR-3, BT-474, MDA-MB-468, MDA-MB-231 (KRas mutant),SUM229PE (KRas mutant), SUM159PT (HRas mutant), and Hs578T (HRas mutant; ref. 40). The characteristics of the cell lines

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 ofAKT 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 EGFfor 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) andAKT signaling inwild-typeMCF-7 cells (CTR cells) and PI4KIIa knockoutMCF-7 cells (KO cells; E). Cellswere treatedwith 1mmol/L PI-273 orDMSO for 24 hours (for lipid ratio detection) or 3 days (for AKT signaling detection), respectively. Lipid contents were detected by the appropriate MassELISA Kit. AKT phosphorylation and protein expression levels were measured by Western blot analysis. All the values are presented as the means� SD from threeindependent experiments, and all the above experiments were performed three times in triplicate. � , P < 0.01; #ns, not significant.

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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 weretreated for 48 hourswith 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 eachcell-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 for48 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-231cells. Anchorage-dependent cell growth and anchorage-independent cell growth were measured using the plate clone-forming assay and the soft agarclone-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 aboveexperiments 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,origin, estrogen receptor (ER), progesterone receptor (PR), humanepidermal growth factor receptor 2 (HER-2), Ras, mice tumori-genicity and the PI-273 IC50 value, are summarized in Supple-mentary Table S3. As shown in Fig. 5A, Ras-mutant breast cancercell lines are less sensitive to PI-273, as the PI-273 IC50 values forMCF-7, T-47D, SK-BR-3, MDA-MB-468, and BT-474 cells were3.5 mmol/L, 3.1 mmol/L, 2.3 mmol/L, 3.9 mmol/L and 2.1 mmol/L,respectively, while the IC50 values for all the Ras-mutant cell lineswere higher than 10 mmol/L. The 56-carboxyfluorescein diacetatesuccinimidyl ester (CFDA SE) assay was used to evaluate theeffects of PI-273 on cell proliferation. As shown in SupplementaryFig. S8A, 1 mmol/L and 2 mmol/L PI-273 inhibited the cellproliferation of both MCF-7 and T-47D cells in a time-dependentmanner, but these PI-273doses did not influence the proliferationofHs 578T cells. These results are consistent with the inhibition ofthese compounds against PI4KIIa in different cell lines andindicate that MCF-7 and T-47D cells are much more sensitive toPI-273. Cell-cycle regulation by PI-273 was examined by 3,8-diamino-5-[3-(diethylmethylammonio) propyl]-6-phenylphe-nanthridinium diiodide staining and flow cytometry. Interesting-ly, PI-273 blocked the cell cycle at the G2–M phase (Fig. 5B),similar to the effect of PIK-75, another substrate-competitiveinhibitor of PIKs (PI3Ka; ref. 41). The effect of PI-273 on cellapoptosis was evaluated by a terminal deoxynucleotidyl transfer-ase dip nick end labeling (TUNEL) assay. As shown in Fig. 5C,treatmentwith PI-273 induced cell apoptosis in all three Raswild-type breast cancer cells: MCF-7, T-47D, and SK-BR-3. In additionto the cell viability assay, the inhibitory effects of PI-273 on thetumorigenicity of cells in vitro were evaluated with plate clone-forming tests (Fig. 5D) and soft agar clone-forming tests (Fig. 5E).PI-273 exhibited prominent drug potency in MCF-7 and T-47Dcells (1 mmol/L PI-273 reduced both plate colony and soft agarcolony growth bymore than 50%). However, Hs 578T andMDA-MB-231 cells were less sensitive to PI-273 thanMCF-7 and T-47Dcells. To assess whether sensitivity is dependent on PI4KIIaexpression in different cell lines, the PI4KIIa expression levels(Supplementary Fig. S8B) and PI4P regulation effects (Supple-mentary Fig. S8C) were measured. The results showed that all thecell lines had high PI4KIIa expression levels, and the PI4P contentcould be regulated by PI-273 in all four cell lines tested. AlthoughthePI4KIIa expression levelswere different among these cell lines,such differences could not distinguish the sensitivity of PI-273.However, the cell sensitivities to PI-273 showed strong correlationwith the Ras status (Fig. 5A–E; Supplementary Table S3). Takentogether, these results indicate that PI-273 retards cell prolifera-tion by blocking cells at the G2–M phase, inducing cell apoptosisand suppressing cell tumorigenicity, and these effects are depen-dent on the cell type.

Antitumor effect of PI-273 in a xenograft modelTo determine whether PI-273 can suppress breast cancer in vivo,

we engraftedMCF-7 cells, which were confirmed to be sensitive toPI-273 in the above experiments, into the right flank region ofBALB/c nude mice. Four days after cell injection, the mice wererandomized and received either an intraperitoneal injection of PI-273 25 mg/kg/day or vehicle (5% DMSO). The mice were sacri-ficed 24 hours after the 15th injection. PI-273 profoundly sup-pressed the tumor volume (Fig. 6A) and weight (Fig. 6B) in theMCF-7 xenografts compared to the effects of treatment with thevehicle. Another group of parallel experiments indicated that an

intraperitoneal injection of PI-273 at 50 mg/kg/2 days alsoinhibited MCF-7 xenograft tumor growth, although with lowerefficiency than the 25 mg/kg/day injection (Supplementary Fig.S9A and S9B). We also assessed the toxic effects of PI-273 inmice,and no lethargy, weight loss (Fig. 6C), or other physical indicatorsof sickness were observed. Various tissues, including liver, intes-tine, lung, kidney, spleen and stomach tissue, were examined byhematoxylin eosin (H&E) staining, and no tissue damage (mac-roscopic ormicroscopic)was observed (Supplementary Fig. S9C).These studies establish the effectiveness and safety of PI-273 forantitumor applications. We further assessed the utility of PI-273in animal experiments. SD ratswere treatedwith 0.5mg/kg PI-273intravenously or 1.5mg/kg PI-273 intragastrically. Blood samplescollected retro-orbitally were collected at 0.08, 0.16, 0.33, 0.67, 1,1.5, 2, 3, and 5 hours after the intravenous and intragastricaladministrations. The plasma levels were analyzed by LC/MS-MS,and the summarized pharmacokinetic results are presentedin Table 1. Compound PI-273 is retained in rats at a half-life of0.411 hours for intravenous administration and 1.321 hours forintragastrical administration, and the absolute bioavailability ofPI-273 is 5.1%. These pharmacokinetic results indicate that PI-273 as lead compound showed moderate pharmacokinetic activ-ities. Histologic examination using the TUNEL assay to detectapoptotic cells revealed a significant increase in the number ofapoptotic cells in the PI-273–treatedMCF-7 xenografts comparedwith those treated with vehicle (Fig. 6D). A significant decrease inproliferation, measured by Ki-67 staining, was observed in the PI-273–treated MCF-7 xenografts compared with the vehicle(Fig. 6E). The PI4P content and p-AKT levels in MCF-7–inducedtumors were measured to evaluate the effect of PI-273 in thexenograft model. Consistent with the data shown above, the PI-273–treated tumors exhibited reduced PI4P content and p-AKTlevels (Fig. 6F). These results confirm that the PI4KIIa-specific SMIPI-273 suppresses proliferation, survival, and AKT signaling inMCF-7–induced breast cancer in vivo. The mechanism underlyingthe PI-273 antitumor effect is summarized in Fig. 6G; PI-273inhibits the kinase activity of PI4KIIa, leading to reduction of theintracellular PI4P content and suppression of the PI3K/AKTsignaling pathways, resulting in retarded cell proliferation andincreased apoptosis, and, consequently, tumor growth inhibition.PI-273 shows good potential for use in clinical trials for humanbreast cancer.

DiscussionBecause PI4KIIa plays important roles in Golgi trafficking (14,

42, 43) and tumor progression (3, 4, 44), SMIs of PI4KIIa havepotential as chemical tools for studying the biological function ofPI4KIIa and breast cancer treatment (10, 19, 42). Here, PI-273was screened out and identified as the most potent inhibitor ofPI4KIIa activity and breast cancer cell viability. Biochemistry andkinetic analyses revealed that PI-273 directly binds and reversiblyinhibits PI4KIIa. The fact that PI-273 is a substrate-competitiveinhibitor rather than an ATP-competitive inhibitor is its greatestadvantage, as this results in kinase isoform selectivity. Furtherstudy confirmed that PI4KIIa is essential for the effects of PI-273.As the first substrate-competitive inhibitor in the PI4K family andthe third such inhibitor in the entire PIK family [PIK-75 is asubstrate-competitive inhibitor of PI3K (23) and NIH-12848 is asubstrate-competitive inhibitor of PI5P4Kg (25)], PI-273 is highlyisoform-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 treatedwith 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 thehistologic sections ofMCF-7 xenografts. The y-axis represents the ratio of TUNELþ cells per ten fields for one tumor sample (n¼ 5 tumors/treatment). Scale bar, 20mm.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 (n¼ 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 inMCF-7 xenograft tumors. All the values are presented as themeans� SD from the indicated samples. � , P < 0.01.G,Model of the antitumor effect of PI-273. PI-273 canspecifically 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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without toxicity, showing its great potential for basic PI4KIIa andpharmaceutical research.

Numerous small molecules have been previously identified asPIK inhibitors, as summarized in Supplementary Table S4, andnone affect PI4KIIa, except for EGCG (45), resveratrol (46), andadenosine (47). In fact, we tested the effects of all the potentialcandidates on PI4KIIa activity prior to screening, includingEGCG, resveratrol, adenosine, PI3K inhibitors (Wortmannin,LY294002, ZSTK474), a PI3Ka inhibitor (PI103), a PI3Kd inhib-itor (IC87114), a PI3Kg inhibitor (AS605240), and a PI4KIIIbinhibitor (PIK93; Supplementary Fig. S10). As observed previ-ously, PI4KIIawas insensitive to PI3K and type III PI4K inhibitors(10, 48, 49), and the IC50 values of both resveratrol and EGCGwere greater than 100 mmol/L. Moreover, these molecules are allbroad-spectrum inhibitors and thus cannot be used for PI4KIIasubtype-specific functional studies. Therefore, screening subtype-specific inhibitors for PI4KIIa is of great significance.

As summarized in Supplementary Table S4, hundreds of smallmolecules were identified as PIK inhibitors by screening, butmostwere ATP-competitive, except for PIK-75 (23) and NIH-12848(25), limiting their potential for high isoform selectivity. The lackof structural information regarding the PIK substrate complex is abarrier to developing substrate-competitive inhibitors becauselipid substrate binding requires the kinase to adopt a mem-brane-bound conformation that is likely different from the struc-ture observed in crystal form. We have recently overcomethis limitation by combining data generated from crystal struc-ture, molecular docking, and biochemical studies to identifythe putative PI-binding pocket of PI4KIIa. We determined thata PI4KIIa-unique insertion, the palmitoylation insertion,affects the conformation of the substrate-binding pocket and thusregulates its kinase activity (26). In this study, we found that PI-273 can directly bind the palmitoylation insertion and functionsas a PI-competitive reversible inhibitor (Figs. 2D, 2E, 3A,and 3B; Table 1). These results also confirm our previous MDsimulation results that the palmitoylation insertion influencessubstrate binding. Our work suggests that MD simulation may behelpful for identifying PIK substrate-binding pockets, thus facil-itating substrate-competitive inhibitor screening. However, withrespect to the PI-competitive mechanism of PI-273, determiningwhether PI-273 can directly bind the substrate pocket or onlyinduce allosteric regulation of the substrate-binding site is diffi-cult because of the lack of inhibitor-soaked crystal structureinformation. Additional efforts to elucidate the binding mecha-nism of PI-273 will be made in the future using NMR, crystal

structure, hydrogen deuterium exchange (HDX), and single-mol-ecule fluorescence resonance energy transfer (FRET) analysis.

In this study, we demonstrated that PI-273 can inhibit breastcancer cell proliferation (Supplementary Fig. S8A), block the cellcycle (Fig. 5B) and induce cell apoptosis (Fig. 5C).We then furtherconfirmed its effects on colony formation (Fig. 5D and E) and onan MCF-7 cell-induced xenograft model (Fig. 6A and B), all ofwhich indicated the suppressive effect of PI-273 on breast cancergrowth both in vitro and in vivo. Because previous studies fromourgroup and others have suggested that in addition to breast cancer,many other cancers, such as malignant melanoma and thyroidcarcinoma, also feature high PI4KIIa expression (4, 16), wesuspect that PI-273 may have inhibitory effects on these types oftumors. In our experiment, we also tested the effects of PI-273 onMDA-MB-435 cells, which have been identified as malignantmelanoma cells (50–52). To our surprise, the MDA-MB-435 cellswere even more sensitive to PI-273 in both the plate clone-forming test and the soft agar clone-forming test comparedwith the MCF-7 and T-47D cells (data not shown), suggestingthat PI-273 may suppress melanoma growth and merits furtherinvestigation.

Because precision medicine is considered the optimal meansof completely conquering cancer, clarifying which patientcategories are sensitive to particular drugs is important. In ourstudy, Hs 578T, MDA-MB-231, SUM229PE, and SUM159PTcells were relatively resistant to PI-273 in terms of cell viability,cell cycle, and apoptosis (Fig. 5), which is consistent withprevious studies concluding that monotherapies targeting thePIK signaling pathway are largely disappointing for Ras-mutant cancers (53). PI3K/AKT and Ras/MEK/ERK are twocrucial interlinked growth and survival signaling pathways intumors (52), and Torbett and colleagues indicated that Hs578T cells with activated forms of the H-Ras oncogene result inthe activation of parallel pathways that are able to compensatefor the loss of PI3K signals. Different groups have indicatedthat PI3K inhibitor treatment alone does not result in theregression of Ras-mutant lung tumors (54, 55), pancreaticcancer (56), or non–small cell lung cancer (57) because ofRas/MEK/ERK–dependent signaling activation. However, com-pared with monotherapies, combining the two treatments(MEK and PI3K inhibitors) resulted in a significant survivaladvantage in these cancers. Taken together, these results sug-gest that the pharmacodynamic effects of PI-273 may also berelated to Ras activity, which is another interesting topic to beexplored in future work.

Interestingly, because PI-273 treatment and PI4KIIa knockouthad negligible effects on the PI(4,5)P2 and PI(3,4,5)P3 levels(Figs. 1D and 4D), we hypothesize that the effect of PI4KIIainhibitors on AKT activity does not result from the direct regu-lation of the PI(4,5)P2 and PI(3,4,5)P3 levels. Instead, numerousstudies have indicated that PI4KIIa is important for receptoractivity via trafficking regulation (13, 17, 58, 59). Therefore, itis possible that the regulation of PI4KIIa inhibitors on AKTactivity is dependent on its receptor regulation effect, but thisrequires further validation.

In summary, we identified PI-273 as a lead substrate-compet-itive inhibitor of PI4KIIa based on a unique insertion, the pal-mitoylation insertion, of PI4KIIa as abinding site.Our results alsodemonstrate the significance, safety, and efficacy of PI4KIIa as atherapeutic target, and PI-273 will be optimized as a therapeuticagent for cancer in the future.

Table 1. Pharmacokinetic results of the SD rats after intravenous administrationof 0.5 mg/kg PI-273 and intragastrical administration of 1.5 mg/kg PI-273,respectively.

PI-273

Pharmacokineticparameters

Intravenously(0.5 mg/kg)

Intragastrically(1.5 mg/kg)

Mean � SD Mean � SD

Cmax (ng*mL�1) 2392.7 � 46 116 � 23Tmax (h) 0.083 � 0.00 0.278 � 0.096t1/2 (h) 0.411 � 0.087 1.321 � 0.657MRT0–12 (h) 0.23 � 0.072 1.25 � 0.061MRT0–¥ (h) 0.23 � 0.072 1.571 � 0.378CL/F (L*h�1

*kg�1) 0.649 � 0.204 11.4 � 1.32Vd/F (L*kg�1) 0.38 � 0.118 21.0 � 7.86AUC0–12 (ng*h*mL�1) 816 � 218 125 � 8.28AUC0–¥ (ng*h*mL�1) 816 � 218 132 � 16.0

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Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Li, Z. Gao, D. Zhao, H. Jiang, C. Luo, C. ChenDevelopment of methodology: J. Li, Z. Gao, L. Zhang, C. LuoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Li, Z. Gao, X. Qiao, Y. Zhao, P. ZhangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Li, Z. Gao, D. Zhao, Y. Zhao,H.Ding, J. Lu,H. Jiang,C. Luo, C. ChenWriting, review, and/or revision of the manuscript: J. Li, Z. Gao, D. Zhao,C. Luo, C. ChenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Li, Z. Gao, D. Zhao, L. Zhang, J. Liu, C. ChenStudy supervision: H. Jiang, C. Luo, C. Chen

AcknowledgmentsWe thank Pietro De Camilli for providing the PI4KIIa antibody and Shane

Minogue for providing the full-length human PI4KIIa cDNA.

Grant SupportThis research was supported by National Key R&D Program of China

(2017YFA0504000, 2016YFC0903100 to C. Chen), Personalized Medicines-Molecular Signature-based Drug Discovery and Development, the StrategicPriority Research Program of the Chinese Academy of Sciences (XDA12020316toC.Chen), theNationalNatural Sciences Foundation ofChina (31570857 and31225012 to C. Chen; 31101021 and 81472839 to J. Li; and 81430084,81625022, and 21472208 to C. Luo), the "863" National High-TechnologyDevelopment Program of China (0A200202D03 to C. Chen), NationalKey Scientific Instrument & Equipment Development Program of China(2012YQ03026010 to C. Luo), NNCAS-2012-2 to C. Chen, the BeijingNatural Science Foundation (7132156 to J. Li), and the Science and TechnologyCommission of Shanghai Municipality (15431903100 to J. Li)

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 22, 2017; revised June 29, 2017; accepted August 14, 2017;published OnlineFirst August 21, 2017.

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, Inhibits the Growth of Breast Cancer Cellsαof PI4KIIPI-273, a Substrate-Competitive, Specific Small-Molecule Inhibitor

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