Development of a PDEδ‐Targeting PROTACs that Impair Lipid Metabolism

Abstract The prenyl‐protein chaperone PDEδ modulates the localization of lipidated proteins in the cell, but current knowledge about its biological function is limited. Small‐molecule inhibitors that target the PDEδ prenyl‐binding site have proven invaluable in the analysis of biological processes mediated by PDEδ, like KRas cellular trafficking. However, allosteric inhibitor release from PDEδ by the Arl2/3 GTPases limits their application. We describe the development of new proteolysis‐targeting chimeras (PROTACs) that efficiently and selectively reduce PDEδ levels in cells through induced proteasomal degradation. Application of the PDEδ PROTACs increased sterol regulatory element binding protein (SREBP)‐mediated gene expression of enzymes involved in lipid metabolism, which was accompanied by elevated levels of cholesterol precursors. This finding for the first time demonstrates that PDEδ function plays a role in the regulation of enzymes of the mevalonate pathway.

Supporting Tables   Table S1: Significantly downregulated proteins. HeLa cells were treated for 5 h or 24 h with 1 µM of PROTAC 3 and were subjected to a proteome-wide analysis. Significant boarders were set to p < 0.00001 (Benjamini-Hochberg corrected).

Cell culture
HeLa and Panc Tu-I cells were grown in Dulbecco's Modified Eagle's Medium (4.5 g/L glucose, 4 mM glutamine) supplemented with 10% fetal bovine serum, 10 mM sodium pyruvate and nonessential amino acids (PAN-Biotech). Jurkat cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and non-essential amino acids (PAN-Biotech). All cell lines were cultivated in a humidified incubator at 37 °C and 5% CO2. Adherent cells were splitted every 2-3 days to never reach confluency higher than 80%. Jurkat cells were maintained in a cell concentration between 1 x 10 5 and 1 x 10 6 viable cells/mL.

Cell lysate preparation
For immunoblotting, cells were washed twice with ice-cold PBS and lysed using 2x Laemmli buffer without reducing agent and bromphenol blue. Obtained lysates were sonicated and protein concentration was determined using DC TM Protein Assay (Bio-Rad). Prior to SDS-PAGE 5% (v/v) DTT and 0.05% (w/v) bromphenol blue was added to each lysate.
For proteome profiling, cells were washed once with ice-cold PBS and trypsinized for 5 min.
Detached cells were washed twice with ice-cold PBS and centrifuged at 500xg for 5 min. The resulting cell suspension was dissolved in 500 µL PBS containing protease inhibitors and subjected to five freeze-thaw cycles. After 15 min at 15000xg at 4 °C, protein concentration was determined using the DC TM assay (Bio-Rad).

Immunoblotting
Cell lysate corresponding to 200 µg protein was subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred onto a PVDF membrane using tank

Live cell imaging of mCherry-PDE
Prior to treatment 2,000 HeLa cells were seeded into 96-well plates. Cells were transfected with an mCherry-PDE expressing plasmid (gift from Philippe I.H. Bastiaens) [4] using Lipofectamine 2000 according to manufacturer´s protocol. After one day, cells were treated with the compound and fluorescence intensity was detected using the IncuCyte ® S3 Live Cell Analysis System.

Establishment of HeLa stably expressing NanoLuc ® -PDE fusion protein
PDE was cloned into the pFN31K vector by using the restriction sites AsiSI and PmeI. Then, cells were transfected with Fugene HD (Promega E4881) using manufacturers protocols. Cells were grown in medium containing 500-800 µg/ml of G418 to induce construct insertion. The selected clone showed constant expression of the fusion protein analysed by wester blotting.

Detection of NanoLuc ® -PDE fusion protein
One day prior to treatment, 2,000 HeLa cells stably expressing nanoLuc-PDE were seeded per well into 96-well plates. After 24 h the medium was exchanged with 100 µL DMEM containing the respective compound. NanoLuc activity was measured after 24 h of compound treatment using the Nano-Glo ® Luciferase Assay System (Promega).

Quantification of metabolites
The quantification of selected lipid metabolites was carried out by The Metabolomics Innovation Centre in Canada (TMIC). One day prior to treatment, 400,000 cells were seeded into a 10 mm dish. Cells were treated for 24 h with 1 µM Deltasonamide 1, washed once with ice-cold PBS and trypsinized. Afterwards cells were centrifuged for 5 min at 300xg and separated from the supernatant. After two washing steps with PBS, supernatants were removed, cells snap frozen in liquid nitrogen and shipped to TMIC on dry ice.

Sample preparation for mass spectrometry
One day prior to treatment 400,000 HeLa cells were seeded into a 10-mm dish. Then, the medium All experiments were performed in biological triplicates.

Mass spectrometry (MS)
Prior to nanoHPLC-MS/MS analysis samples were fractionated into 10 fractions on a C18 column using high pH conditions to reduce the complexity of the samples and thereby increasing the number of quantified proteins. Therefore, samples were dissolved in 120 μl of 20 mM ammonium formate (NH4COOH) at pH 11, followed by incubation in an ultra-sonicator for 2 min, subsequent vortexing for 1 min and centrifugation at 8,000xg for 3 min at room temperature. 50 μl of the  The mass accuracy for full mass spectra was set to 20 ppm (first search) and 4.5 ppm (second search), respectively and for MS/MS spectra to 20 ppm. The false discovery rates for peptide and protein identification were set to 1%. Only proteins for which at least two peptides were quantified were chosen for further validation. Relative quantification of proteins was carried out using the reporter ion MS2 algorithm implemented in MaxQuant.
The proteinGroups.txt file was used for further analysis. All proteins which were not identified with at least two razor and unique peptides in at least one biological replicate were filtered off. For further data analysis the "Reporter intensity corrected" corresponding to compound treatment was divided by the "Reporter intensity corrected" of the corresponding vehicle control and the results were written into a new column. This file was stored under a different file name in txt-format. For further data analysis Perseus version 1.6.2.3 was used. [6] The calculated ratios of the abovementioned file were defined as main columns. Proteins resulting from the reverse database search, just identified by site, typical contaminants and not quantified in at least three out of three or four replicates, respectively, were filtered off. The ratios of the "Reporter intensities corrected" were logarithmized (log2) and normalized to the median. The mean of the replicates was calculated and the outlier test "Significance A" was performed. Proteins with a p-value < 0.00001 were considered as statistically highly significantly up-or down-regulated, depending on the direction of change. Proteins that were up-or downregulated, respectively, by other, PDEunrelated Pomalidomide-based PROTACs were removed from the lists. For graphical representation the p-value was logarithmized (-log10) and plotted against the normalized log2value of the mean of reporter ion intensity corrected of the compound divided by the mean of the reporter ion intensity corrected of the DMSO vehicle control.

Cloning, expression and purification of recombinant proteins
The gene encoding Cereblon319-442 was PCR amplified from pAJ075_hsCRBN vector.
The insert was subcloned into the BamHI and SalI restriction sites of the pGEX-6p-2rbs vector for bacterial expression with a N-terminal, PreScission-cleavable GST tag. The plasmid was verified by DNA sequencing.

Chemical Synthesis General information
All reactions were carried out in heat dried glassware under argon atmosphere. All commercially available compounds were used as provided without further purifications.
The ester (2.6 g, 3.35 mmol, 1 eq.) was used in the next reaction without any purification step in between. The compound was dissolved in 40 ml of a mixture of THF/H2O (2:1) and LiOH (802 mg, 30 mmol, 10 eq.) was slowly added. After 16 h at room temperature the solvent was removed, and the residue was dissolved in cold acetonitrile. The suspension was filtered to afford the pure product