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  • Functional characterization of an isoform-selective inhibitor of PI3K

    p110β as a potential anti-cancer agent

    Ni et al, Supplementary data

    Supplementary Methods

    Pharmacokinetic evaluation of KIN-193.

    This analysis was performed at the Sai Advantium Pharma Limited Company (India) with 3 groups

    of 9 male Swiss Albino mice. Animals in Group 1 were dosed via the tail vein at 1 mg/kg of KIN-

    193 solution (7.5% v/v N-methyl pyrrolidone and 40% v/v polyethylene glycol-400 in normal

    saline). Group 2 animals were dosed orally at 10 mg/kg with a suspension formulation (0.5% w/v Na

    CMC with 0.1% v/v Tween-80 in water) of KIN193. Group 3 animals were dosed intraperitoneally

    at 10 mg/kg with a formulation of KIN-193 in 10% v/v ethanol and 20% v/v PEG-400 in water.

    Blood samples were collected at 0, 0.08 (for iv and ip only), 0.25, 0.5, 1, 2, 4, 6, (for oral and ip

    only) 8, 12 and 24 hours (for iv, oral and ip). The blood samples were collected from sets of three

    mice at each time point in tubes containing K2EDTA as an anticoagulant. Plasma samples were

    separated by centrifugation and stored below -70°C until bioanalysis. All samples were processed by

    precipitation using acetonitrile and analyzed with a LC-MS/MS method (LLOQ - 1.138 ng/mL).

    Pharmacokinetic parameters were calculated using the non-compartmental analysis tool of

    WinNonlin® Enterprise software (version 5.2).

    Supplementary Figure legends:

    Figure S1. Establishment of a cell-based system for PI3K isoform-specific inhibitor screening.

    HMEC cells stably expressing myristoylated-HA-tagged p110α (myr-p110α, termed CA-p110α),

    myr-p110β (CA-p110β) or myr-p110δ (CA-p110δ) individually were serum-starved for 2 hours,

  • then treated with PIK-75 (p110α inhibitor), TGX-221 (β inhibitor), IC87751 (δ inhibitor) or GDC-

    0941 (pan inhibitor) at the indicated doses for 1h. Cell lysates were prepared and subjected to

    western blot assays with the indicated antibodies.

    Figure S2. Identification of KIN-193 as a p110β specific inhibitor. A, Schematic flow chart of

    Lanthascreen cellular assay of plate-based fluorescence-screening for testing of compounds’

    inhibition of AKT phosphorylation at both T308 and S473 in HMEC cell lines expressing CA-

    p110α, CA-p110β or CA-p110δ. B, IC50 values for selected small molecule inhibitors against various

    PI3K p110 isoforms in isogenic HMEC lines expressing CA-p110α, CA-p110β, or CA-p110δ using

    Lanthascreen assays.

    Figure S3. HMEC cell lines stably expressing CA-p110α, CA-p110β or CA-p110δ were serum-

    starved for 2 hours, then treated with TGX-221 or KIN-193 at the indicated doses for 1h. Cell lysates

    were prepared and subjected to western blot assays with the indicated antibodies.

    Figure S4. Western blot analyses of PTEN and PI3K protein expression in the indicated cell lines.

    Figure S5. Pharmacokinetics of KIN-193 in mice. A, Plasma concentrations of KIN-193 following

    a single administration IV (1 mg/kg), PO (10 mg/kg), or ip (10 mg/kg) to male Swiss albino mice.

    Plasma samples were analyzed for KIN-193 by LC-MS/MS methods. B, Nude mice bearing ~500

    mm3 Rat-CA-p110α/β xenografts were given a single ip administration of KIN-193 (10 mg/kg).

    Plasma samples were collected at 0, 1, 4, 8, and 24 h following compound administration and

    analyzed for KIN-193 by LC-MS/MS methods.

  • Supplementary Table Titles

    Table S1. Chemical structures of the compounds collected for PI3K isoform-specific cellular assays.

    Table S2. KinomeScanTM profiling of KIN-193 against 433 kinases.

    Table S3. IC50 values for KIN-193 in 422 cancer cell lines. The PTEN status is given for each cell

    line.

  • Ni et al Supplementary Figure. 1

    C A

    -p 11

    0α c

    el ls

    D M

    S O

    co nt

    ro l c

    el ls

    PIK-75

    0.05 0.1

    TGX-221 IC87114

    0.01 0.1 0.5 0.01 0.1 0.5 0.01

    GDC-0941

    0.1 0.5 0.01

    pAktS473

    pAktT308

    Akt

    α-tubulin

    pAktS473

    pAktT308

    Akt

    α-tubulin

    pAktS473

    pAktT308

    Akt

    α-tubulin C A

    -p 11

    0β c

    el ls

    C

    A -p

    11 0δ

    c el

    ls

    p110 inhibitor α β δ pan

    Dose (µM)

  • (Serum free)

    add inhibitor BacMam GFP-Akt

    HMEC- CA-p110α CA-p110β CA-p110δ

    overnight overnight

    plate cells Lyse cells in

    presence of Tb- pAkt-Ab

    1h

    detection

    2h

    LS-pAkt308 (HMEC-CAp110b-BacAkt)

    -4 -2 0 2 0

    2

    4

    6

    8

    10

    [TGX-221] (uM)

    R e

    sp n

    o se

    R a

    tio

    IC50

    TGX-221

    0.02487IC50= 0.025 µM Response ratio: 8.3 Z’ factor: 0.78

    log[TGX-221] (µM)

    A

    B

    Ni et al Supplementary Figure. 2

    CAp110α- pAktT308

    CAp110α- pAktS473

    CAp110β- pAktT308

    CAp110β- pAktS473

    CAp110δ- pAktT308

    CAp110δ- pAktS473

    KIN-150 0.2038 0.0135 2.1155 0.0217 0.3544 0.0210

    KIN-151 0.0253 0.0393 0.1769 0.2342 0.1796 0.1574

    KIN-152 0.0183 0.0254 0.2861 0.2629 0.0109 0.0119

    KIN-167 0.0136 0.0092 0.0068 0.0059 0.0007 0.0003

    KIN-169 > 5 > 5 0.0169 0.0206 0.2471 0.3101

    KIN-170 0.4256 0.3122 0.4957 0.5554 0.3339 0.2067

    KIN-171 0.0206 0.0121 0.0111 0.0096 0.0104 0.0086

    KIN-172 0.0801 0.0585 0.0948 0.0797 0.0689 0.0581

    KIN-173 0.2872 0.3081 1.6980 2.5220 1.1210 0.8935

    KIN-174 0.7453 0.8733 1.9150 2.0270 0.3356 0.5583

    KIN-193 > 5 > 5 0.0117 0.0224 0.8206 0.9566

    KIN-194 > 5 > 5 > 5 > 5 0.4959 0.8307

    KIN-195 0.5918 0.9826 > 5 > 5 0.2490 0.2523

    KIN-196 > 5 > 5 1.2462 1.7370 0.0078 0.0213

    KIN-197 0.0146 0.0160 0.0289 0.0332 0.0066 0.0081

    KIN-203 0.0076 0.0119 0.2952 0.1601 0.0033 0.0052

    KIN-266 0.0280 0.0033 0.5455 0.0029 0.1032 0.0075

    KIN-267 0.0616 0.0820 0.2239 0.2064 0.0242 0.0308

    KIN-268 0.1213 0.0322 0.7398 0.0327 0.1500 0.0553

  • D M

    S O

    C on

    tro l c

    el ls

    pAktS473

    pAktT308

    Akt

    C A

    -p 11

    0α c

    el ls

    α-tubulin

    TGX-221

    0.1 1 0.01 Dose (µM)

    KIN-193

    0.1 1 0.01 C

    A -p

    11 0β

    c el

    ls

    pAktS473

    pAktT308

    Akt

    α-tubulin

    Ni et al Supplementary Figure. 3

    C A

    -p 11

    0δ c

    el ls

    pAktS473

    pAktT308

    Akt

    α-tubulin

  • p85

    vinculin

    p110α

    p110β

    PTEN

    Ni et al Supplementary Figure. 4

  • Route Cmax (ng/mL) Tmax (h) AUCINF

    (hr*ng/mL) T1/2 (hr) Cl

    (mL/min/Kg) Vss (L/Kg) F (%)

    IV(1mg) 780 - 112 2.75 148 10.3 - PO(10mg) 74.1 0.25 101 - - - 6.85 I.P.(10mg) 2074 0.08 1140 1.1 - - -

    A

    B

    [K IN

    -1 93

    ] ( µM

    )

    Time (hr)

    blood

    0h 1h 4h 8h 24 h

    0

    2

    4

    6

    [KIN-193]

    Ni et al Supplementary Figure. 5

  • Supplementary table 1

    KIN150 BEZ235 CC(C)(C#N)C1=CC=C(C=C1)N2C3=C4 C=C(C=CC4=NC=C3N(C2=O)C)C5=C C6=CC=CC=C6N=C5

    Chemistry 1 Mol Cancer Ther. 2008 Jul;7(7):1851-63. Epub 2008 Jul 7. Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3- kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity. Maira SM, Stauffer F, Brueggen J, Furet P, Schnell C, Fritsch C, Brachmann S, Chène P, De Pover A, Schoemaker K, Fabbro D, Gabriel D, Simonen M, Murphy L, Finan P, Sellers W, García-Echeverría C.

    KIN151 CN(S(=O)(C1=CC([N+]([O- ])=O)=CC=C1C)=O)/[N]([H])=C/C2=C N=C3N2C=C(Br)C=C3.[Cl]

    Chemistry 2 Bioorg Med Chem. 2007 Sep 1;15(17):5837-44. Epub 2007 Jun 6. Synthesis and biological evaluation of sulfonylhydrazone-substituted imidazo[1,2-a]pyridines as novel PI3 kinase p110alpha inhibitors. Hayakawa M, Kawaguchi K, Kaizawa H, Koizumi T, Ohishi T, Yamano M, Okada M, Ohta M, Tsukamoto S, Raynaud FI, Parker P, Workman P, Waterfield MD.

    KIN152 O=C(C1=CC=CN=C1)NC2=NC3=C(OC )C(OC)=CC=C3C4=NCCN24

    Chemistry 3 Cancer Cell. 2006 May;9(5):341-9. A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma. Fan QW, Knight ZA, Goldenberg DD, Yu W, Mostov KE, Stokoe D, Shokat KM, Weiss WA.

    KIN167

    ZSTK474

    FC(F)C1=NC2=C(C=CC=C2)N1C3=NC (N4CCOCC4)=NC(N5CCOCC5)=N3

    Chemistry 4 J Natl Cancer Inst. 2006 Apr 19;98(8):545-56. Antitumor activity of ZSTK474, a new phosphatidylinositol 3-kinase inhibitor. Yaguchi S, Fukui Y, Koshimizu I, Yoshimi H, Matsuno T, Gouda H, Hirono S, Yamazaki K, Yamori T.

    KIN169 TGX-221 CC(C=C1C(C)NC2=CC=CC=C2)=CN3 C1=NC(N4CCOCC4)=CC3=O

    Chemistry 5 Nat Med. 2005 May;11(5):507-14. Epub 2005 Apr 17. PI 3-kinase p110beta: a new target for antithrombotic therapy. Jackson SP, Schoenwaelder SM, Goncalves I, Nesbitt WS, Yap CL, Wright CE, Kenche V, Anderson KE, Dopheide SM, Yuan Y, Sturgeon SA, Prabaharan H, Thompso