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Purity: ≥98%
PF‑06446846 (PF06446846; PF‑6446846) is a novel and potent PCSK9 (proprotein convertase subtilisin/kexin type 9) inhibitor that inhibits the translation of PCSK9 by inducing the ribosome to stall around codon, mediated by the sequence of the nascent chain within the exit tunnel. PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. PF-06446846 is highly selective for the inhibition of PCSK9 translation. Inhibiting PCSK9 can potentiate CD8 + T cell antitumor activity by promoting LDLR-mediated TCR recycling and signaling. Thus PCSK9/LDLR may serve as a potential target for cancer immunotherapy as well.
| Targets |
Human 80S ribosome (Kd = 7.0 μM in saturation binding assay). [1]
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| ln Vitro |
PF-06446846 suppresses Huh7 cells from secreting PCSK9, with an IC50 of 0.3 μM[1]. PCSK9(1-35)-luciferase expression is inhibited by PF-06446846, with an IC50 of 2 μM[1]. Rat bone marrow and human CD34+ toxicity are demonstrated by PF-06446846 (compound 7f) [2].
PF-06446846 inhibited the secretion of PCSK9 from Huh7 human hepatoma cells with an IC50 of 0.3 μM. [1] In a HeLa-derived cell-free translation system, PF-06446846 inhibited the translation of a PCSK9(1-35)-luciferase fusion protein with an IC50 of 2 μM, while translation of luciferase alone was only inhibited by 20% at 250 μM. [1] Ribosome profiling in Huh7 cells treated with PF-06446846 (1.5 μM, 1 hour) demonstrated highly selective inhibition of PCSK9 translation, with ribosome stalling centered around codon 34 of the PCSK9 transcript. Only 22 other cellular transcripts showed significant sensitivity to PF-06446846 at this timepoint. [1] Stable isotope labeling with amino acids in cell culture (SILAC) proteomic analysis of Huh7 cell secretome and lysates treated with PF-06446846 (0.25 μM and 1.25 μM for 4 or 16 hours) confirmed high selectivity, showing no general effect on the secreted or intracellular proteome aside from PCSK9 and a few other targets. [1] Metabolic labeling of Huh7 cells with ³⁵S-Met/Cys showed that PF-06446846 did not cause global inhibition of protein synthesis, unlike cycloheximide. [1] Activity was dependent on the amino acid sequence of the nascent PCSK9 peptide (residues 1-35), not the mRNA codon sequence. Key sensitive regions included Leu15-Leu20, Trp-containing residues 9-11, and residues 31-33. [1] PF-06446846 inhibited translation in cell-free systems derived from rabbit reticulocytes, wheat germ, and yeast, but not from Escherichia coli. [1] Ribosomal toeprinting assays confirmed that PF-06446846 induced ribosome stalling during translation of PCSK9, with reverse transcriptase termination products consistent with stalling on and after codon 35. [1] Sucrose density gradient analysis of cell-free translation reactions programmed with PCSK9 mRNA showed that PF-06446846 treatment led to the appearance of polysome-associated, radiolabeled small peptides, consistent with a stalled ribosome followed by queued ribosomes. [1] |
| ln Vivo |
In vivo, PF-06446846 lowers total plasma cholesterol and circulating PCSK9 levels without appearing to be harmful [1].
Oral administration of PF-06446846 to male rats at doses of 5, 15, and 50 mg/kg/day for 14 days resulted in dose-dependent lowering of plasma PCSK9 levels following single and repeated dosing. [1] At the 50 mg/kg dose, a statistically significant reduction in total plasma cholesterol (30% decrease) and LDL cholesterol (58% decrease) was observed after 14 days, with no significant change in HDL cholesterol. [1] The reduction in plasma PCSK9 and cholesterol was achieved without modulating plasma markers of liver function (ALT, AST, albumin) and without histopathological findings in the liver. [1] |
| Enzyme Assay |
A saturation binding assay was conducted to measure the binding of [³H]-PF-06446846 to purified human 80S ribosomes. Varying concentrations of the titrated radioligand were incubated alone (for total binding) or with a 200-fold excess of unlabeled PF-06446846 (for nonspecific binding) with 0.6 μM of ribosomes in assay buffer. After incubation, the reaction mixture was filtered, and bound radioactivity was counted. Specific binding was calculated by subtraction, and Kd and Bmax values were determined by fitting the data to a one-site specific binding model. [1]
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| Cell Assay |
Cytotoxicity assay[2]
Cell Types: rat myeloid lineage(−) cells and CD34+ cells Tested Concentrations: 0-20 µM Incubation Duration: 72 hrs (hours) Experimental Results: demonstrated cytotoxicity against rat Lin(−) with an IC50 value of 2.9 µM and 2.7 µM and human CD34+, respectively. For PCSK9 secretion inhibition: Huh7 cells were seeded in 96-well plates, allowed to attach, then treated with PF-06446846 or vehicle in fresh media overnight. Conditioned media was collected, and PCSK9 levels were measured using a commercial human PCSK9 ELISA kit. [1] For metabolic labeling: Huh7 cells were washed and incubated in methionine/cysteine-free medium for 30 minutes. PF-06446846, cycloheximide, or vehicle was added, followed by ³⁵S-Met/Cys labeling mix for 30 minutes. Cells were lysed, and lysates were analyzed by SDS-PAGE and phosphorimaging to quantify ³⁵S incorporation. [1] For SILAC proteomics: Huh7 cells were grown in SILAC media containing light, medium, or heavy isotopic forms of lysine and arginine for several doublings. Cells were synchronized and then treated with vehicle (light), 0.25 μM PF-06446846 (medium), or 1.25 μM PF-06446846 (heavy) for 4 or 16 hours. Conditioned media (secretome) and cell lysates were collected separately. Proteins were digested with trypsin, and peptides were analyzed by LC-MS/MS for identification and relative quantification. [1] |
| Animal Protocol |
Animal/Disease Models: Male SD (SD (Sprague-Dawley)) (Crl:CD [SD] rats, 5 per group; 6-8 weeks old at start of dosing) [1]
Doses: 5, 15, and 50 mg/kg Route of Administration: Every Daily oral administration for 14 days. Experimental Results: Reduce plasma PCSK9, plasma total cholesterol and LDL-C (low-density lipoprotein cholesterol) in a dose-dependent manner without obvious toxicity. Male Sprague-Dawley rats (5 per group) were administered PF-06446846 orally at doses of 5, 15, or 50 mg/kg/day for 14 consecutive days. The compound was formulated in a vehicle of 200 mM citrate buffer in 0.5% methylcellulose (w/v). The control group received vehicle only. The dose volume was 5 mL/kg based on individual body weight. [1] Blood samples were collected on days 1 and 12 at approximately 1, 3, 6, and 24 hours post-dose for measurement of plasma PF-06446846 and PCSK9 concentrations. [1] On day 15 (24 hours after the last dose), fasted animals were humanely killed and necropsied. Terminal blood samples were collected for clinical chemistry (including cholesterol panels) and hematology. The liver was weighed, and a selected set of tissues was collected for histopathological examination. [1] |
| ADME/Pharmacokinetics |
Plasma concentrations of PF-06446846 were determined in rats after oral administration at doses of 5, 15, and 50 mg/kg. Supplementary table (S11) provides concentration data at 1, 3, 6, and 24 hours after administration on days 1 and 12. Specific pharmacokinetic parameters (e.g., half-life, clearance, oral bioavailability) are not reported in the text. [1]
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| Toxicity/Toxicokinetics |
In a 14-day rat study, PF-06446846 was well tolerated at doses of 5 and 15 mg/kg/day, with no dose-limiting changes observed histologically or clinically compared to the excipient. [1]
At a dose of 50 mg/kg/day, the following adverse reactions were observed: a slight decrease in food intake (11-13%), a slight decrease in bone marrow cell count (mainly erythroid cells) associated with a 9% decrease in total red blood cells, a slight decrease in white blood cell (52%), lymphocyte (54%), T cell and B cell counts, and mild necrosis of ileal crypt cells in one of the five animals. [1] No histopathological changes were observed in the liver at any dose. No significant changes were observed in liver transaminase (ALT, AST) or albumin levels. [1] |
| References |
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| Additional Infomation |
PF-06446846 is a triazolopyridine compound with the structure 3H-[1,2,3]triazolo[4,5-b]pyridine, substituted at position 3 with 4-{(3-chloropyridin-2-yl)[(3R)-piperidin-3-yl]carbamoyl}phenyl. It is a potent inhibitor of PCSK9. It has the effects of lipid-lowering drugs and EC 3.4.21.61 (kexin) inhibitors. It is a monochloropyridine compound belonging to the piperidine, tertiary carboxamide, benzamide, and triazolopyridine classes. PF-06446846 is an orally effective small molecule compound that inhibits PCSK9 synthesis by directly targeting the human ribosome during translation. It induces ribosome arrest during the PCSK9 translation elongation phase, specifically near codon 34 after the signal peptide. This mechanism depends on the amino acid sequence of the nascent peptide chain within the ribosome exit channel. [1] Ribosomal profiling analysis showed that although PF-06446846 acts on the core translation mechanism, it is highly selective for PCSK9 and affects only a very small number of other transcripts. [1] This study is the first to demonstrate a small molecule inhibitor of eukaryotic mRNA translation selectivity for therapeutic purposes. However, the narrow boundary between the PCSK9-reducing efficacy and hematopoietic/gut toxicity observed at the highest dose in rats may hinder the clinical development of PF-06446846 itself. [1]
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| Molecular Formula |
C22H20CLN7O
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|---|---|
| Molecular Weight |
433.893502235413
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| Exact Mass |
433.141
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| CAS # |
1632250-49-7
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| Related CAS # |
PF-06446846 hydrochloride;1632250-50-0
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| PubChem CID |
86271238
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
3.1
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
31
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| Complexity |
617
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1C[C@H](CNC1)N(C2=C(C=CC=N2)Cl)C(=O)C3=CC=C(C=C3)N4C5=C(C=CC=N5)N=N4
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| InChi Key |
FDTXHWQFIXYHCL-QGZVFWFLSA-N
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| InChi Code |
InChI=1S/C22H20ClN7O/c23-18-5-2-12-25-20(18)29(17-4-1-11-24-14-17)22(31)15-7-9-16(10-8-15)30-21-19(27-28-30)6-3-13-26-21/h2-3,5-10,12-13,17,24H,1,4,11,14H2/t17-/m1/s1
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| Chemical Name |
(R)-4-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-N-(3-chloropyridin-2-yl)-N-(piperidin-3-yl)benzamide
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| Synonyms |
PF‑06446846; PF-6446846; PF6446846; PF 06446846; PF06446846;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3047 mL | 11.5237 mL | 23.0473 mL | |
| 5 mM | 0.4609 mL | 2.3047 mL | 4.6095 mL | |
| 10 mM | 0.2305 mL | 1.1524 mL | 2.3047 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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