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Mice (8 per group) were injected i.p. with vehicle or 20 mg/kg .... 12 (0.70 g, 1.86 mmol) was dissolved in 10 mL THF/H2O (1:1) and cooled to 0°C for 5min. ..... 1H), 6.85 – 6.69 (m, 3H), 6.63 – 6.55 (m, 2H), 6.51 (s, 1H), 5.50 (dd, J = 8.2, 5.2 Hz, ...

Supplementary Information

Selective inhibitors of the FK506-binding protein 51 by induced fit

S. Gaali1†, A. Kirschner1†, S. Cuboni1 , J. Hartmann1, C. Kozany1,2, G. Balsevich1, C. Namendorf1, P. Fernandez-Vizarra1, C. Sippel1, A.S. Zannas1,3, R. Draenert4, E.B. Binder1, O.F.X. Almeida1, G. Rühter5, M. Uhr1, M.V. Schmidt1, C. Touma1, A. Bracher6, F. Hausch1*

1

Max Planck Institute of Psychiatry, 80804 Munich, Germany

2

Present address: Sequiserve, 85591 Vaterstetten, Germany

3

Department of Psychiatry and Behavioral Sciences, Duke University Medical Center,

27710 NC, USA 4

Medizinische Klinik und Poliklinik IV, Hospital of the University of Munich, 80336

Munich, Germany 5

Lead Discovery Center GmbH, 44227 Dortmund, Germany

6

Max Planck Institute of Biochemistry, 82152 Martinsried, Germany



These authors contributed equally

1

Nature Chemical Biology: doi:10.1038/nchembio.1699

SUPPLEMENTARY RESULTS

Supplementary Table 1| Data Collection and Refinement Statistics Dataset

FKBP51iFit1

FKBP51iFit4

FKBP52iFit-FL

4TW6

4TW7

4TW8

P212121

P21212

P212121

a, b, c (Å)

45.0, 48.6, 56.7

49.3, 60.9, 38.1

45.7, 46.4, 310.1

, ,  (°)

90, 90, 90

90, 90, 90

90, 90, 90

Wavelength (Å)

1.00000

0.97931

0.99998

Resolution (Å)

44.99 - 1.4 (1.47 - 1.4)*

38.32- 1.25 (1.32 – 1.25)

46.35 - 3.0 (3.17 - 3.0)

0.058 (0.535)

0.046 (0.406)

0.143 (0.759)

I/σI

20.5 (3.5)

15.8 (2.3)

10.2 (2.0)

Completeness (%)

99.8 (98.7)

92.4 (63.7)

97.8 (91.0)

7.0 (6.9)

3.5 (2.5)

6.0 (4.7)

Resolution (Å)

20 - 1.4

20 - 1.25

30 – 3.0

No reflections

23835

28336

12971

0. 151/ 0. 186

0.129 / 0.174

0.210 / 0.257

1007

1008

3698

Ligand/ion

58

58

142

Water

194

215

1

Protein

13.03

11.92

54.92

Ligand/ion

11.80

11.41

81.05

Water

27.67

29.86

22.80

0.028

0.022

0.006

Bond angles (°) 2.601 * Values in parenthesis for outer shell.

2.209

1.157

PDB entry Space group Cell dimensions

Rmerge

Redundancy Refinement

Rwork / Rfree Number of atoms Protein

B-factors

R.m.s. deviations Bond length (Å)

2

Nature Chemical Biology: doi:10.1038/nchembio.1699

Supplementary Table 2

A) Blood-Brain Permeability Potential of SAFit1 and SAFit2

Test Recovery Direction Compound (%)

Papp (10-6 cm/s)

A-to-B

88

0.1

B-to-A

91

14.1

A-to-B

30

7.21

B-to-A

49

21.3

SAFit1

SAFit2

Efflux Ratio

Brain Penetration Classification

135

Low

3

Moderate

B) PK parameters of SAFit1 and SAFit2 after injection of 10mg/kg i.p.

SAFit1

SAFit2

t1/2 [h]

2.5

9.7

Tmax [h]

1.0

0.3

Cmax [ng/ml]

1383.0

2094.8

AUC 0-t [h*ng/ml]

6811.4

7117.0

AUC 0-inf_obs [h*ng/ml]

6815.0

8148.1

Vz/F_obs [l/kg]

5.3

17.2

Cl/F_obs [l/h/kg]

1.5

1.2

brain:plasma ratio [%]

< 0.5

16.7

3

Nature Chemical Biology: doi:10.1038/nchembio.1699

Supplementary Figure 1

67

F

FKBP51 and FKBP52 have almost identical FK506-binding sites Superimposed co-crystal structures of the FK506-binding domains of FKBP51 (red: 3O5R, orange: 4DRI, pink: 4DRK) and FKBP52 (marine: 4LAX, blue: 4DRJ, cyan: 4LAY) in complex with FK506

3, 4

, rapamycin5 or a synthetic analog (ligands not shown)1, 4. Active site residues

are highlighted as sticks. Residue Phe67, which was mutated to accommodate the engineered ligand 1, is indicated.

4

Nature Chemical Biology: doi:10.1038/nchembio.1699

Supplementary Figure 2 220 200 180

mPs

160 140 120 100 80 60 1e-6

FKBP51wt FKBP52wt FKBP51F67V FKBP52F67V 1e-5

1e-4

1e-3

1e-2

1e-1

1e+0

1e+1

1e+2

1e+3

Ligand [µM]

Ligand 1 potently binds FKBPF67V mutants but not the wildtype counterparts Affinity of ligand 1 for the purified FK506-binding domains of FBKP51wt, FKBP51F67V, FKBP52wt, FKBP52F67V determined by fluorescence polarization using 40-fluorescein-Glyrapamycin as a tracer6.

5

Nature Chemical Biology: doi:10.1038/nchembio.1699

Supplementary Figure 3 A

FKBP51 and FKBP52 inversely regulate neurite outgrowth (A) N2a cells were transfected with a myr-Venus plasmid together with the indicated FKBPexpressing constructs or empty vector. After two days of serum withdrawal, cells were fixed, imaged for Venus fluorescence and analyzed by ImageJ. (B) Inhibition analysis of N2a cells co-expressing FKBP51 and FKBP52. Under conditions, where FKBP51 and FKBP52 balance each other, dual inhibition of these two FKBPs has no effect. Data represent averages of >30 cells. Error bars indicate ± s.e, ***: p