Volume 23, Issue 11, Pages 1362-1371 (November 2016) A Small-Molecule Anti-secretagogue of PCSK9 Targets the 80S Ribosome to Inhibit PCSK9 Protein Translation  Donna N. Petersen, Julie Hawkins, Wanida Ruangsiriluk, Kimberly A. Stevens, Bruce A. Maguire, Thomas N. O’Connell, Benjamin N. Rocke, Markus Boehm, Roger B. Ruggeri, Tim Rolph, David Hepworth, Paula M. Loria, Philip A. Carpino  Cell Chemical Biology  Volume 23, Issue 11, Pages 1362-1371 (November 2016) DOI: 10.1016/j.chembiol.2016.08.016 Copyright © 2016 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2016 23, 1362-1371DOI: (10. 1016/j. chembiol Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Schematic Representation and Summary of PCSK9 Secretion High-Throughput Screen (A) Representation of the PCSK9 protein including the signal peptide (SP), prodomain (Pro), serine protease catalytic domain (Catalytic), and a C-terminal cysteine/histidine (Cys/His)-rich domain. Amino acid numbering indicates the domain boundaries. (B) Diagram of high-throughput screen (HTS) assay. Recombinant PCSK9-ProLabel (PCSK9-PL) was stably expressed in CHO-K1 cells under a cytomegalovirus promoter (PCMV). Auto-cleavage of the precursor PCSK9-PL results in separation of the prodomain (Pro) from the catalytic domain. The prodomain subsequently re-associates with the catalytic domain to yield mature PCSK9-PL which is secreted by the cell. Secreted recombinant PCSK9-PL levels are measured from conditioned media via β-galactosidase enzyme fragment complementation using the PathHunter EFC Kit (DiscoverX) as depicted. (C) HTS hit triage workflow resulting in the identification of R-IMPP, with the number of compounds selected at each step. R-IMPP activity at the various steps is indicated. SEAP, secreted alkaline phosphatase. Cell Chemical Biology 2016 23, 1362-1371DOI: (10.1016/j.chembiol.2016.08.016) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Analysis of R-IMPP and S-IMPP Activity in Human Hepatoma Cells (A) Quantitation of secreted PCSK9 and transferrin from conditioned media of Huh7 cells treated with vehicle (0.5% DMSO), R-IMPP (30 μM), S-IMPP (30 μM), or fluvastatin (1 μM) for 16–20 hr. Data were normalized to the vehicle control for each experiment and represent the mean ± SD from four separate experiments performed in triplicate. (B) Dose-response curves for inhibition of PCSK9 and statin-induced PCSK9 secretion by R-IMPP. Dotted reference lines represent control levels of PCSK9 after DMSO vehicle (black) or 1 μM fluvastatin (Fluv) treatment (gray). Data are representative of three independent experiments. Error bars reflect the mean ± SD of three replicates. (C) Immunoblot analysis of cellular PCSK9 and LDL-R showing R-IMPP-driven suppression of both pro-PCSK9 (pro) and mature PCSK9 (m) from Huh7 cell lysates and concomitant increases in LDL-R levels upon overnight treatment with R-IMPP. Data are representative of at least three independent experiments. (D) Alexa Fluor-labeled LDL-C (AF-LDL) uptake measured in Huh7 cells after overnight treatment with a dose response of R-IMPP (black) or S-IMPP (light gray). PCSK9 protein (25 μg/mL) and blocking EGF-A peptide (100 μM) were included to demonstrate the responsiveness of the system to PCSK9 levels. Data were normalized to the vehicle control for each experiment and represent mean ± SD from three independent experiments. **p < 0.01, ***p < 0.001 relative to vehicle control as indicated. Cell Chemical Biology 2016 23, 1362-1371DOI: (10.1016/j.chembiol.2016.08.016) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 R-IMPP and S-IMPP Inhibit Binding or Activity of Multiple Control Ligands in a Representative Panel of 113 G Protein-Coupled Receptors, Enzymes, and Transporters R-IMPP and S-IMPP were profiled at 10 μM. Data were generated by Cerep (http://www.cerep.fr/Cerep/Users/index.asp). Colors in the heatmap represent different ranges of inhibitory activities. See also Table S2. Cell Chemical Biology 2016 23, 1362-1371DOI: (10.1016/j.chembiol.2016.08.016) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Characterization of R-IMPP Cellular Activity Shows Rapid Effects on Protein Production (A) Time course of the effects of R-IMPP on PCSK9 secretion. WT-7 cells expressing recombinant V5 epitope-tagged PCSK9 were treated for the indicated times with R-IMPP (30 μM). Cellular PCSK9 and β-actin levels were determined by western blot using anti-V5 and anti-β-actin antibodies, respectively. Data are representative of three independent experiments. (B) Pulse-chase experiment. WT-7 cells were pretreated with R-IMPP (30 μM) for 30 min, followed by a 30 min pulse of 35S-methionine/35S-cysteine. Conditioned media and lysates were collected at 1, 2, and 3 hr post-pulse and subjected to immunoprecipitation with anti-PCSK9 and anti-albumin antibodies. PCSK9 and albumin levels were determined by autoradiography. (C) Test of protease inhibitors. WT-7 cells overexpressing recombinant PCSK9 were pretreated for 2 hr with DMSO vehicle or either a serine protease inhibitor (aprotinin, 2 μg/mL), a cysteine protease inhibitor (ALLN, 17 μg/mL), a lysosomal protease inhibitor (chloroquine, 25 μM), or a proteasome inhibitor (MG132, 1 μM). R-IMPP (30 μM) was then added for 1 hr and cells were harvested for western blot analysis for PCSK9 (anti-V5) and β-actin levels. See also Figures S2 and S3. Cell Chemical Biology 2016 23, 1362-1371DOI: (10.1016/j.chembiol.2016.08.016) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Structure/Function Studies Show that R-IMPP Inhibition Is Dependent on Sequences within the Signal Sequence and Prodomain (A) Schematic of full-length PCSK9 and domain deletion constructs for structure-function studies. All constructs for transient transfection were engineered with a C-terminal V5 tag for immunodetection. (B) Effects of R-IMPP and S-IMPP treatment on production of full-length PCSK9 and various truncated mutants. PCSK9 expression constructs were transfected into Huh7 cells, followed by treatment with DMSO or R-IMPP (30 μM) or S-IMPP (30 μM). After 24 hr, cells were harvested and analyzed for cellular PCSK9 levels (anti-V5) and β-actin via western blot. (C) Schematic of luciferase fusion constructs and prodomain scanning deletion mutants. (D) Constructs were transiently transfected into Huh7 cells and luciferase reporter activity was assessed after treatment with vehicle (0.1% DMSO), R-IMPP (30 μM), or S-IMPP (30 μM). Luciferase activity was normalized for transfection efficiency via co-transfection of a β-galactosidase reporter. Data represent the mean ± SD of three replicates. An ANOVA with post hoc Dunnett test was conducted for each deletion (SAS 9.4) with a significant level at *p = 0.05. Cell Chemical Biology 2016 23, 1362-1371DOI: (10.1016/j.chembiol.2016.08.016) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 R-IMPP Inhibits PCSK9 Translation and Binds Directly to 80S Ribosomes (A) Schematic of in vitro translation reporter constructs encoding full-length PCSK9-luciferase (PCSK9-FL), prodomain-luciferase (1–152), or luciferase alone (Luc). (B) Luciferase activity of HeLa-based cell-free translation reactions programmed with mRNAs encoding PCSK9-FL, (1–152), or Luc in the presence of R-IMPP (30 μM) or S-IMPP (30 μM). Data were normalized to the vehicle (DMSO) control for each experiment and represent mean ± SD from three independent experiments. ***p < 0.001 relative to vehicle control. (C) Overlaid extracted ion chromatograms of test compounds and controls (10 μM each) from ribosomal binding experiments after incubation with either human (7.6 μM, top panel) or E. coli (7.0 μM, bottom panel) ribosomes, followed by ASMS. Data are representative of three separate injections. Note, conclusions on relative binding affinity cannot be implied from the ASMS ion chromatogram traces due to differences in ionization efficiencies between R-IMPP/S-IMPP and homoharringtonine. Relative R-IMPP and S-IMPP peak heights can be compared due to the expectation that enantiomers will ionize with the same efficiency. Cell Chemical Biology 2016 23, 1362-1371DOI: (10.1016/j.chembiol.2016.08.016) Copyright © 2016 Elsevier Ltd Terms and Conditions