The identification of AF38469; an orally bioavailable inhibitor of the VPS10P family sorting receptor Sortilin

Tenna Juul Schrødera, Søren Christensena, Samsa Lindberga, Morten Langgårda, Laurent Davida, Philip J. Maltasa, Jørgen Eskildsena, Jan Jacobsena, Lena Tagmosea, Klaus Bæk Simonsena, Lars Christian Biilmann Rønna, Inge de Jonga, Ibrahim J Malika, Jens-Jakob Karlssona, Jan Egebjergb, Jeffrey B. Stavenhagena, Dorthe Strandbygårdb, Søren Thirupb, Jacob Lauwring Andersenb, Srinivas Uppalanchic, Sridhar Pervaramc, Shiva Prasad Kasturic, Pradheep Eradic, Durga Rao Sakumudic, Steve P Watsona.a Neuroscience Drug Discovery, H- Lundbeck A/S, Copenhagen, Denmark;b The Lundbeck Foundation Research Centre MIND, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, Aarhus C, 8000, Denmark; c Medicinal Chemistry, GVK BioScience, Hyderabad, India

Supplementary Data

Methods

Sortilin was crystallized as previously described (Andersen, 2013, submitted) with a 10 molar excess of

AF438469 for 1 hour. 1 μl of sortilin-AF38469 was mixed with a 1 μl reservoir and equilibrated against 500 μl

reservoir using the sitting-drop vapour-diffusion method at 292 K in 24-well crystallization plates. The best

diffracting crystals were obtained in 0.1 M HEPES-Tris pH 7.3, 0.4 M sodium malonate, 27 % (w/v) PEG 3350

(Hampton Research) and 4.5 % (v/v) glycerol. The crystals grew to a size of 200 x 100 x 50 μm over two weeks.

Crystals were dehydrated by addition of 100 μl 80 % (v/v) PEG400 to the reservoir and mounted in Litholoops

(Molecular Dimensions) from the mother liquor, excess mother liquor was dipped away by gently touching the

side of the drop well with the edge of the loop 1 and flash cooled in liquid N2. A complete single-wavelength (λ =

0.9 Å) data set of 3600 oscillation images, with 0.1o oscillation was collected at 100 K on the X06DA (PXIII)

beamline at the Swiss Light Source (SLS) using the PILATUS 2M-F detector (Dectris). The diffraction images

were processed in XDS 2 and scaled in SCALA 3. Molecular replacement was performed with the program

PHASER 4 using a search model derived from the structure of sortilin in complex with neurotensin (PDB ID

3F6K) 5. Rigid body refinement, generation of ligand coordinates and restraints, calculation of omit maps and

refinement were performed in PHENIX 6. Model building and analysis was performed using Coot 7. The final

model quality was analysed using Molprobity 8. Superpositions were performed and structural figures were

prepared using PyMol 9.

Supplementary table 1. Data-collection and refinement statistics for the sortilin-AF38469 complex.

Values in parentheses are for the highest resolution shell (2.7 – 2.85Å)

†Rmerge=∑hkl

∑i

|I i (hkl )−⟨ I (hkl ) ⟩|/∑hkl

∑i

I i (hkl ), where I i (hkl ) is the intensity of the ith observation and

⟨ I (hkl ) ⟩ is the mean intensity of the reflection hkl. §

Rp . i. m=∑hkl

[1/ (N−1)]1/2∑i|I i (hkl )− ⟨ I (hkl ) ⟩|/∑

hkl∑

iI i (hkl ), where N is the multiplicity of a given reflection.

#R=∑h

||Fobs|−|Fcalc||/∑h

|Fob s|, where Fobs and F calc are the observed and calculated structure factors

respectively.

Data Collection

Space groupUnit-cell parameters (Å, o)

Resolution range (Å)No. of unique reflectionsRmerge

†

Rp.i.m§

I/σ(I)Completeness (%)MultiplicityIsotropic B-factor (Wilson)

RefinementResolution range (Å)No. of refelctionsRwork / Rfree

#

No. of atomsProtein (non-H)AF38469GlycosylationsWaterB factors (Å2)ProteinAF40431GlycosylationsWaterR.m.s. deviationsBond lengths (Å)Bond angles (o)Ramachandran plotMost favouredAllowed regionOutlier regionPDB code

C2a = 161.9, b = 77.4,c = 111.2 , β = 127.1o

44.4 – 2.7303977.1 (84.5)2.9 (36.0)15.2 (2.2)99.7 (97.9)6.984.3

44.4 – 2.73036620.1 / 24.2

5140236751

92.872.793.7.664.4

0.0111.354

97.82.20.04N7E

Supplementary figure 1. Electron density maps of AF38469. A) Bias reduced simulated annealing Fo – Fc map

contoured at 3.0 level. B) Final 2Fo – Fc map contoured at 1.5 level.

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