Human 80S ribosome (binds selectively; no IC50/Ki/EC50 reported). [1]

R-IMPP increases LDL-R levels without altering the quantities of released transferrin, hence stimulating the uptake of LDL-C by liver cancer cells. R-IMPP causes transcription-dependent inhibition of PCSK9 translation but neither increases nor decreases PCSK9 transcription. R-IMPP binds to human ribosomes only, not to those of E. Coli [1].

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In Huh7 cells, R-IMPP (30 μM) decreased secreted PCSK9 levels and increased cell-surface LDL-R, leading to increased uptake of Alexa Fluor 546-labeled LDL (47.5% ± 6.6% increase). No effect on transferrin secretion. [1]
In WT-7 cells (Huh7 overexpressing V5-tagged PCSK9), R-IMPP (30 μM) decreased PCSK9 protein levels within 1 hour, without affecting PCSK9 mRNA (qPCR). Pulse-chase experiment showed decreased production of pro-PCSK9 and mature PCSK9 with no effect on albumin. Proteasome (MG132, ALLN) and lysosomal (chloroquine, aprotinin) inhibitors did not reverse R-IMPP effect, ruling out increased degradation. [1]
R-IMPP inhibited translation of full-length PCSK9-luciferase and prodomain (1-152)-luciferase fusion mRNAs in HeLa cell-free in vitro translation assays (30 μM), but did not inhibit translation of luciferase alone. [1]
R-IMPP bound selectively to human 80S ribosomes but not E. coli ribosomes as shown by affinity-selection mass spectrometry; S-IMPP showed much weaker binding. [1]

For ribosome binding assay (ASMS): Purified human (7.6 μM) and E. coli (7.0 μM) ribosomes were incubated with test compounds (10 μM each) and subjected to affinity-selection mass spectrometry. Homoharringtonine and azithromycin were used as binding controls for human and E. coli ribosomes, respectively. Overlaid extracted ion chromatograms were recorded. [1]
For ribosome purification: Ribosomes from HeLa cells and E. coli were purified as described previously (Maguire et al., 2008; Khatter et al., 2014). Quality was established by analytical ultracentrifugation and protein mass spectrometry. [1]

For high-throughput phenotypic screen: CHO-K1 cells overexpressing ProLabel-tagged PCSK9 were used. Hits were evaluated in multiple cellular toxicity assays and counterscreens for effects on other secreted proteins. [1]
For PCSK9 and transferrin ELISA (AlphaLISA): Huh7 cells were plated at 7,500 cells/well in 384-well plates with 2 μL test compound. Final volume 20 μL. Cells incubated overnight (16-20 hr) at 37°C, 5% CO2. Then 5 μL of biotinylated antibody (3 nM final) and antibody-conjugated Alphabeads (10 μg/mL final) added for 0.5 hr, followed by 5 μL streptavidin donor beads (40 μg/mL final) for 1 hr at room temperature. Read on EnVision plate reader (Ex 680 nm, Em 615 nm). [1]
For LDL uptake assay: Huh7 cells seeded at 20,000 cells/well in black 96-well clear-bottom plate. Serum-free medium with DMSO or treatments overnight, then incubated with 10 μg/mL AF-LDL for 24 hr. Cells washed with TBS+2 mg/mL BSA, lysed with RIPA buffer. Fluorescence read at Ex 520 nm, Em 580 nm. LDL uptake normalized to protein concentration (bicinchoninic acid assay). [1]
For LDL-R immunoblot: Protein lysates separated on 4%-12% SDS-PAGE, transferred to nitrocellulose, blocked with 3% non-fat dry milk, incubated with primary antibody (goat anti-human LDL-R, rabbit anti-PCSK9, mouse anti-β-actin), then HRP-conjugated secondary antibody, signal detected with chemiluminescence. [1]
For qRT-PCR: Detailed in Supplemental Experimental Procedures (not fully described in main text). [1]
For time course: WT-7 cells plated at 1×10^6 cells/well in 6-well dishes, treated with vehicle or 30 μM R-IMPP for 0.5,1,2,4,8,24 hr. Cells lysed in 2× SDS-PAGE buffer. PCSK9 and β-actin detected by immunoblot using anti-V5 antibody (1:5000) and anti-β-actin. [1]
For protease treatment: WT-7 cells pretreated for 2 hr with 2 μg/mL aprotinin, 17 μg/mL ALLN, 25 μM chloroquine, or 1 μM MG132, then 30 μM R-IMPP for 1 hr. PCSK9 and β-actin levels determined by immunoblot. [1]
For pulse-chase: WT-7 cells pretreated with 0.1% DMSO or 30 μM R-IMPP for 2 hr in methionine/cysteine-free RPMI, then 35S-labeled amino acids for 0.5 hr. Chase for 1,2,3 hr in complete media with DMSO or R-IMPP. Cell lysates and media immunoprecipitated with anti-PCSK9 or anti-albumin antibody, separated on SDS-PAGE, dried, and exposed for phosphoimaging. [1]
For PCSK9 truncation studies: Huh7 cells transfected with full-length or truncated PCSK9-V5 constructs (1 μg DNA, except 2 μg for PCSK9-152) using Fugene 6. After 24 hr, treated with vehicle, 30 μM R-IMPP, or 30 μM S-IMPP for 24 hr. Lysates immunoblotted for V5 and β-actin. [1]
For luciferase reporter assay: DNA encoding PCSK9 signal sequence and prodomain fused to firefly luciferase (pCMV-PCSK9proLuc) and 20-amino acid deletion mutants were transiently transfected into Huh7 cells. After 24 hr, treated with vehicle, 30 μM R-IMPP, or 30 μM S-IMPP for 24 hr. Luciferase activity normalized to β-galactosidase co-transfection. Data represented as mean ± SD of three replicates. [1]
For in vitro translation assay: HeLa lysate prepared from suspension cells. Linear template DNA transcribed to mRNA using T7 kit. 50 nL test compound added to 384-well plate with 0.5% DMSO vehicle and 50 μM puromycin as control. 10 μL IVT mix containing HeLa lysate, 1.25 mM ATP, 0.12 mM GTP, 20 mM creatine phosphate, 60 μg/mL creatine phosphohexane, 90 μg/mL tRNA, amino acids, 1.6 mM magnesium acetate, 112 mM potassium acetate, 4.6 mM TCEP, 8 units RNasin, and 0.1 μg luciferase reporter mRNA. Incubated at 30°C for 45 min. Then 1 μL transferred to 25 μL Steady-Glo and read on EnVision plate reader. [1]

Molecular weight: 389. LogD (pH 7.4): 1.1. Permeability (Papp AB in MDCKII-LE cells): 5.4 × 10^-6 cm/s. [1]

Cytotoxicity in THLE cells (ATP depletion, 72 hr): IC50 = 230 μM. [1]
Caspase-Glo 3/7 apoptosis assay: IC50 > 300 μM. [1]
HepG2 cells (72 hr): IC50 = 79 μM. [1]
HepG2 cells in glucose (24 hr): IC50 = 160 μM. [1]
HepG2 cells in galactose (24 hr): IC50 = 100 μM. [1]

[1]. A Small-Molecule Anti-secretagogue of PCSK9 Targets the 80S Ribosome to Inhibit PCSK9 Protein Translation. Cell Chem Biol. 2016 Nov 17;23(11):1362-1371.

R-IMPP is the first small-molecule anti-secretagogue of PCSK9 that acts by inhibiting PCSK9 translation via binding to human 80S ribosomes. Its activity is sequence-dependent, requiring the signal peptide and part of the prodomain. It does not affect general protein synthesis (no effect on luciferase alone or albumin). The enantiomer S-IMPP is inactive in PCSK9 secretion and LDL uptake assays. R-IMPP binds selectively to human but not E. coli ribosomes. This represents a novel mechanism for modulating PCSK9. [1]

C24H27N3O2

389.490085840225

389.21

C, 74.01; H, 6.99; N, 10.79; O, 8.22

2133832-83-2

2133832-83-2;2173005-10-0 (HCl);

122198706

Light yellow to yellow solid powder

3.8

1

4

6

29

520

1

COC1=CC=C(C=C1)CCC(=O)N([C@@H]2CCCNC2)C3=NC=CC4=CC=CC=C43

(R)-N-(isoquinolin-1-yl)-3-(4-methoxyphenyl)-N-(piperidin-3-yl)propanamide

OKTGXQMSOIQCTJ-HXUWFJFHSA-N

R-IMPP RIMPP R IMPP

R-IMPP PF-00932239 RIMPP PF00932239 R IMPP

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~256.75 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.42 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (6.42 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.42 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)