an inhibitor of the proteasomal deubiquitinating …10.1074/jbc.m117.815126/...cell death (2,3). b,...

1

AninhibitoroftheproteasomaldeubiquitinatingenzymeUSP14induces taueliminationinculturedneurons

MonicaBoselli1§,Byung-HoonLee1,2§,JessicaRobert1,MiguelA.Prado1,Sang-won

Min3,ChialinCheng4,M.CatarinaSilva4,ChanghyunSeong1,5,SuzanneElsasser1,KetkiM.Hatle1,TimothyC.Gahman6,StevenP.Gygi1,StephenJ.Haggarty4,LiGan3,Randall

W.King1*andDanielFinley1* SUPPLEMENTAL DATA FILES SUPPL. TABLE LEGEND SUPPL. TABLE 1 SAR analysis of IU1. Structure-activity relationship (SAR) analysis of the initial 37 synthesized analogs of IU1. Compounds were tested for their potency and selectivity for USP14 over IsoT/USP5 by Ub-AMC hydrolysis assay as described in the EXPERIMENTAL PROCEDURES. Percent inhibition was measured at 8 µM; these values represent means of duplicate measurements. IC50 values were determined in triplicate for compounds more potent than IU1.

SUPPL. TABLE 2 SAR analysis of IU1. Structure-activity relationship (SAR) analysis of 50 synthesized analogs of IU1. These compounds were designed using IU1-47 as a scaffold, in contrast to those of SUPPL. TABLE 1, which are based on IU1. Percent inhibition was measured at 4 µM and IC50 values were determined for compounds more potent than IU1. SUPPLEMENTAL FIGURE LEGENDS

SUPPL. FIGURE 1 Analytical NMR of IU1-47. 1H-NMR spectroscopic data of IU1-47. Proton integration is included above each peak. Chemical shifts for each peak are displayed across the top of the spectrum.

SUPPL. FIGURE 2 Analytical LC-MS of IU1-47. LC-UV-MS analysis of IU1-47 in positive ion mode. A, the compound was analyzed on a C18 reverse phase column over an acetonitrile gradient measuring UV absorbance at 254 nm. B, total ion count. C, shared peak count extracted from the peak with retention time at 3.5 min. The observed purity was >95% and the molecular weight 331.1 Da. SUPPL. FIGURE 3 LC-MS analysis of IU1-47 stability by USP14. A, IU1-47 (2 µM) was incubated in the presence or absence of USP14 (2 µM). Proteasome was then added at 0.5 µM to activate USP14. After 50 min at room temperature, the reaction mixture was applied to an LC-MS analytical system (Agilent series 1200LC/6130MS) with a reverse-phase pentafluorophenyl (PFP) column (Phenomenex). The total ion count of each LC-MS trace (m/z at 331) was monitored and the peak area integrated. The graph represents the mean of triplicate measurements, with error bars indicating standard deviations. B and C, each representative LC-MS chromatogram of IU1-47 with or without USP14 incubation. The retention time for IU1-47 is ~8.0 min. X-axis represents retention time (min) and Y-axis total ion count.

2

SUPPL. FIGURE 4 The enhancement of tau degradation by IU1-47 in murine embryonic fibroblasts is mediated by USP14. Wild-type and Usp14-/- MEFs were transfected with a pcDNA3.1-derived plasmid expressing full length, untagged human-tau 2N4R, then treated with IU1-47 for 9 hrs. Samples were prepared and immunoblots were probed for total tau (tau5) and GAPDH. Control Usp14-/- MEFs transfected with the same tau construct and subjected to 9 hr of IU1-47 treatment do not show decreased tau levels.

SUPPL. FIGURE 5 Dose-dependence cytotoxicity of IU1-47 in MEFs. Usp14-/- and wild-type MEFs were treated for 24 hr with increasing concentrations of IU1-47 (ranging from 3.2 – 562 µM) and cell viability was analyzed using the MTT assay as previously done for IU1 (1). Dose-response curves were fitted using nonlinear regression [log(inhibitor) vs response - variable slope] modeled with GraphPad Prism 5 to obtain the EC50 values. Values correspond to the average of two independent experiments. Error bars represent SEM. Note that IU1-47 is more toxic in Usp14-/- MEFs than in WT MEFs, indicating that the toxicity of the compound, which is seen only at high doses, is off-target; whereas the effect on tau elimination appears to be on-target.

SUPPL. FIGURE 6 IU1-47 treatment in human iPSC-derived neurons. A, viability of neural progenitor cell (NPC) line derived through an iPSC intermediate from human fibroblasts was assayed for cell viability using CellTiter-Glo after treatment with IU1-47 at the indicated concentrations for 48 hr. Treatment with 1 µM Staurosporine was used as a positive control for cell death (2,3). B, Neurons derived in vitro from NPCs through a six-week program of terminal differentiation (DIV 42) were treated with the indicated concentrations of IU1-47 for 96 hr, then assayed for cell viability using Toxilight bioassay (Lonza). C, DIC images of neurons derived in vitro from NPCs for six weeks and treated with 30 µM IU1-47 and DMSO-vehicle. The results show that 96-hr treatment with IU1-47 does not induce significant morphological changes as compared to the DMSO-vehicle treated neurons. SUPPL. FIGURE 7 Cytotoxicity comparison based on quantitation of intracellular ATP using CellTiter-Glo® assay of cortical primary neurons cultured in either DMEM or in Neurobasal. A, Rat cortical primary neurons were plated and cultured in Neurobasal medium for 6 days. Medium was then switched to either Neurobasal (fresh) or DMEM in presence of the indicated treatments for 24 hr (CellTiter Glo® Viability Assay, which measures ATP content). Error bars represent SD (n=2 samples from 1 experiment). B, Mouse cortical neurons (DIV4) were incubated with graded doses of IU1-47 for 48 hr. Cells were harvested and lysates prepared. Proteins were resolved by SDS-PAGE and immunoblotted using a monoclonal antibody against total tau (tau5). GAPDH was used as a loading control.

REFERENCES FOR SUPPLEMENTAL MATERIAL

1. Lee, B.-H., Lee, M. J., Park, S., Oh, D.-C., Elsasser, S., Chen, P.-C., Gartner, C., Dimova, N., Hanna, J., Gygi, S. P., Wilson, S. M., King, R. W., and Finley, D. (2010) Enhancement of proteasome activity by a small-molecule inhibitor of USP14. Nature 467, 179–184

2. Belmokhtar, C. A., Hillion, J., and Ségal-Bendirdjian, E. (2001) Staurosporine induces apoptosis through both caspase-dependent and caspase-independent mechanisms. Oncogene 20, 3354–3362

3. Wiesner, D. A., and Dawson, G. (1996) Staurosporine Induces Programmed Cell Death in

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Embryonic Neurons and Activation of the Ceramide Pathway. J. Neurochem. 66, 1418–1425

Enhancement of proteasome function in primary neurons

4

SUPPLEMENTAL TABLE 1

SAR analysis of IU1-1 to IU1-46

Name Compound

Percent inhibition IC50

USP14/26S IsoT USP14/26S IsoT

IU1

(from screen)

70 10 5.5 100

IU1

(resyn-thesized)

74 6 3.9 80

IU1-1

36 0

IU1-2

82 7 1.7 55

IU1-3

53 0


5

Name Compound



IU1-4

55 0

IU1-5

0 0

IU1-6

0 0

IU1-7

52 2

IU1-8

19 0

IU1-14

20 11

IU1-15

44 10


6

Name Compound



IU1-16

0 2

IU1-17

21 11

IU1-18

41 8

IU1-19

0 4

IU1-24

1 6

IU1-25

0 13

IU1-26

0 7

N

OHN


7

Name Compound



IU1-27

0 8

IU1-28

20 10

IU1-29

7 7

IU1-30

3 8

IU1-31

4 7

IU1-32

24 6

IU1-33

92 27 1.1 33

NF

ON


8

Name Compound



IU1-34

29 6

IU1-35

29 4

IU1-36

0 4

IU1-37

0 1

IU1-38

45 2

IU1-39

3 1

IU1-40

46 8


9

Name Compound



IU1-41

0 0

IU1-42

5 0

IU1-43

84 22 1.9 34

IU1-44

93 25 0.7 25

IU1-45

5 0

IU1-46

70 5 9.7 93


10

SUPPLEMENTAL TABLE 2

SAR analysis of IU1-47 to IU1-96

Name Compound



IU1-47

92 18 0.6 20

IU1-48

31 0

IU1-49

49 0

IU1-50

61 0

IU1-51

69 5

IU1-52

57 12

NCl

ON

NCl

ON

O

NCl

ON

N

N

ON

Cl

O

NCl

ON

NCl

ON


11

Name Compound



IU1-53

63 11

IU1-54

85 11 1 29

IU1-55

60 8

IU1-56

78 11

IU1-57

36 8

IU1-58

36 7

IU1-59

60 10

NCl

OHN

NCl

ON

O

NCl

ON

NCl

ON

NCl

ON

O

NCl

ON

N

NCl

ON

N


12

Name Compound



IU1-60

89 9 0.8 31

IU1-61

87 16 0.8 18

IU1-62

53 2

IU1-63

55 6

IU1-64

48 0

IU1-65

16 0

IU1-66

20 0

NCl

ON

NO2N

ON

NO2N

ON

NO2N

ON

NO

ON

NO

ON

NO

ON


13

Name Compound



IU1-67

4 3

IU1-68

7 2

IU1-69

86 18 3.2 53

IU1-70

4 5

IU1-71

9 8

IU1-72

17 5

N

ON

N

ON

NCl

ON

F

NClNH

O

NClNH

O

NClNH

ON


14

Name Compound



IU1-73

0 0

IU1-74

50 2

IU1-75

77 16 0.9 24

IU1-76

4 1

IU1-77

8 1

IU1-78

69 0

IU1-79

49 0

N

ON

N

NN

Cl

ON

NI

ON

NClNH

ON

NN3

O

N

Cl

O

NO

N

Cl

O

N

O


15

Name Compound



IU1-80

71 0

IU1-81

52 3

IU1-82

68 15

IU1-83

52 6

IU1-84

34 0

IU1-85

5 6

N

Cl

O

N

N

Cl

O

O

NH

N

Cl

O

O

N

N

Cl

O

N

N

Cl

O

NO

N

S

O

N

H2N

O O


16

Name Compound



IU1-86

4 0

IU1-87

63 11

IU1-88

73 7

IU1-89 NCl

ON

82 8

IU1-90 NCl

ON

73 7

IU1-91 NCl

ON

24 0

NO

N

H2N

O

N

Cl

O

NO

NCl

ON

N


17

Name Compound



IU1-92 NCl

ON

9 0

IU1-93 NCl

ON N

13 2

IU1-94 NCl

ON

F

86 18

IU1-95 NCl

ON

Cl

74 15

IU1-96 NCl

ON

O

40 16

SupplementalFigure1Enhancement of proteasome function in primary neurons

SF1

PPM

SignalIntensity

SupplementalFigures

SupplementalFigure2Enhancement of proteasome function in primary neurons

SF2

Time(min)

Time(min)

B

A

m/z

C

B

C

A

SupplementalFigure3

IU1-47

IU1-47+USP14

IU1-47 IU1-47+USP14

%TotalionCo

unt


SF3 RetenEonEme(min)

RetenEonEme(min)

Totalion

cou

nt

Totalion

cou

nt

IU1-47(µM)

+/+MEFs Usp14-/-MEFspcDNA3.1-Tau(2N4R)

GAPDH

Totaltau(tau5)

40 500+

20+ + +

0-

0 20 40 50+ + + +

0-

65

37


SF4

SupplementalFigure4

10 100 10000

50

100

150 WT MEF(EC50 159 µM)

Usp14-/- MEF(EC50 96 µM)

[IU1-47] µM

Cel

l Via

bilit

y (%

)Enhancement of proteasome function in primary neurons

SF5

SupplementalFigure5

A

C

IU1-47(30µM)DMSO

iPSC-derivedneurons

0

50

100

150

%viablecells

NeuralProgenitorCells

SupplementalFigure6%deadcells

0

50

100

BiPSC-derivedneurons


SF6

SupplementalFigure7

B

DM

EM

DM

SO

DM

EM

st a

ur o

sp

or i n

e

NB

B2

7 D

MS

O

NB

B2

7 s

t au

r os

po

r i ne

0

5 0

1 0 0

'

% v

iab

ilit

y

D M E M D M S O

D M E M s t a u r o s p o r i n e

N B B 2 7 D M S O

N B B 2 7 s t a u r o s p o r i n e

100

50

0

%viablecells DMEMStaurosporine

DMEMDMSO

NBB27DMSONBB27Staurosporine

A

Tau(clone5)

IU1-47(µM)0 2.5 5 7.5 10 12.5

GAPDH

50

37

252015

75

SF7

an inhibitor of the proteasomal deubiquitinating …10.1074/jbc.m117.815126/...cell death (2,3). b,...

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