Supplementary Material for the Manuscript ... Supplementary Material for the Manuscript A novel...

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  • Supplementary Material for the Manuscript

    A novel multi-modal RAGE-specific inhibitor controls amyloid-β-mediated brain disorder in mice

    Rashid Deane1*, Itender Singh1*, Abhay P. Sagare1, Robert D. Bell1, Nathan T. Ross2,3, Barbra LaRue1, Rachal Love1, Sheldon Perry1, Nicole Paquette1, Richard J. Deane1, Thiyagarajan Meenakshisundaram1, Troy Zarcone3, Gunter Fritz5, Alan E. Friedman4, Benjamin L. Miller2,3, Berislav V. Zlokovic1,5. 1Center of Neurodegenerative and Vascular Brain Disorders, 2Department of Biochemistry and Biophysics, 3Department of Dermatology, 4Department of Environmental Medicine, University of Rochester, Rochester, NY, USA, and 5Department of Neuropathology, University of Freiburg, Freiburg, Germany, and 5Center for Neurodegeneration and Regeneration at the Zilkha Neurogenetic Institute and Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. Correspondence: Berislav V. Zlokovic, M.D., Ph.D. Center for Neurodegeneration and Regeneration The Zilkha Neurogenetic Institute Keck School of Medicine, University of Southern California 1501 San Pablo St. Los Angeles, CA 90089 Email: berislav_zlokovic@urmc.rochester.edu *First co-authors

    mailto:berislav_zlokovic@urmc.rochester.edu

  • 100 2000

    0.2

    0.4

    0.6

    A β4

    0 bi

    nd in

    g

    (n m

    ol es

    /1 04

    c el

    ls )

    A

    Aβ40 (nM)

    RAGE

    4824 24

    - +pcDNA3-RAGE

    Time (h)

    +

    β-Actin

    RAGE-CHO cells

    pcDNA3-RAGE

    pcDNA3-GFP

    B

    0 5 50 125 500 1000 10000

    50

    25

    0

    125

    75

    100

    (nM)

    C el

    l s ur

    vi va

    l (%

    o f c

    on tr

    ol )

    C RAGE-CHO cellsRAGE-CHO cells

    pcDNA3-GFP -+ -

    Supplementary Figure 1. Saturable Aβ binding to RAGE-CHO cells. (A) Western blot analysis of RAGE levels in RAGE-transfected CHO cells (pcDNA3-RAGE; RAGE-CHO cells) and mock-transfected CHO cells (pcDNA3-GFP, green fluorescent protein). (B) 125I-Aβ40 binding to RAGE-CHO cells (closed squares) and mock-transfected CHO cells (closed triangles) in the presence of various concentrations of unlabeled Aβ40. (C) CHO cell survival with and without the indicated concentrations of FPS2 (gray column) or FPS-ZM1 (black column). Values are mean ± s.e.m., n = 3 independent experiments.

  • N

    B

    A C

    O A NH2 B H

    O

    C Cl

    O +

    NaBH3CN (1.0 eq)

    (1.0 eq) (1.0 eq) MeOH / EtOH 64 deg. C, 9 h B

    HN A

    +

    (1.1 eq)

    DMAP (0.1 eq) DIPEA (1.1 eq)

    dry CH2Cl2 RT, 30 min

    A = hydrophobic amine B = electron rich aromatic C = electron poor benzene

    Hydrophobic Amines

    Electron Rich Aromatic Group

    Electron Deficient Benzene

    N1 N2 N3

    B1 B2 B3 B4 B5

    NH2

    N4

    NH2 NH2 NH2

    H

    O H

    O H

    O

    N H

    O H

    F

    H

    O

    Cl

    Cl

    O

    Cl Cl

    Cl

    O

    N+ O-O

    N+ O-

    O Cl

    O

    N+ O-O

    Cl

    O

    N+ O-O

    Cl

    O

    Cl

    A1 A2 A3 A4 A5

    Supplementary Figure 2. Diagram for the synthesis of the second generation library of compounds. The overall reaction equation is shown at the top. The various substitutes of hydrophobic amines (N1 to N4), electron aromatic groups (A1 to A5) and electron deficient benzene (B1 to B5) are shown below the overall reaction equation. DMAP (4-N,N- dimethylpyridine); DIPEA (diisopropylethylamine). For details see the Results section.

  • Supplementary Figure 3. FPS-ZM1 binds to immobilized sRAGE but not to immobilized Aβ40. (A) FPS-ZM1 was detected in the pellet containing immobilized sRAGE (left), but not in the supernatant containing no sRAGE (right). (B) FPS-ZM1 was absent in the pellet containing immobilized Aβ40 (left) but present in the supernatant containing no Aβ40 (right).

    0 2 4 6 8 10 Time (min)

    A B

    F P

    S -Z

    M 1

    in te

    ns ity

    x 1

    0 3 /

    sa m

    pl e

    Immobilized sRAGE Supernatant

    0

    20

    40

    60

    F P

    S -Z

    M 1

    in te

    ns ity

    x 1

    0 3 /

    sa m

    pl e

    0

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    40

    60

    0 2 4 6 8 10 Time (min)

    0 2 4 6 8 10 0

    40

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    160

    0 2 4 6 8 10 0

    40

    80

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    160

    Time (min)Time (min)

    F P

    S -Z

    M 1

    in te

    ns ity

    x 1

    0 3 /

    sa m

    pl e

    F P

    S -Z

    M 1

    in te

    ns ity

    x 1

    0 3 /

    sa m

    pl e

    Immobilized Aβ Supernatant

  • Aβ40 − + + +

    N uc

    le ar

    N F

    -κ B

    re

    la tiv

    e ab

    un da

    nc e

    P

  • P

  • 0

    2

    4

    6

    8

    10

    (f m

    ol /m

    im /g

    IS F

    ) p

  • Vehicle

    FPS2

    FPS-ZM1

    0

    25

    50

    75

    100

    D H

    E fl

    uo re

    sc en

    ce in

    n eu

    ro ns

    (% o

    f v eh

    ic le

    -t re

    at ed

    c on

    tr ol

    s)

    Vehicle

    FPS-ZM1 FPS2

    -

    + - +

    -

    -

    + - -

    P

  • − + +

    +− −

    NF-κB

    Free Probe

    BV-2 cells

    − + +

    +− −

    0

    3

    6

    9 P