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Purity: ≥98%
PF-06928215 (PF06928215) is a novel, potent, high affinity and selective cGAS (cyclic GMP-AMP Synthase) inhibitor with the potential to be used for autoimmune diseases and inflammatory conditions. It inhibits cGAS with an IC50 of 4.9 μΜ. PF-06928215 is a high affinity (KD = 200 nM) active site-targeting, substrate-competitive cyclic GMP-AMP synthase (cGAS) inhibitor (IC50 = 4.9 μM; in the presence of 1 mM ATP, 0.3 mM GTP, 100 nM 45-bp interferon-stimulatory dsDNA (ISD)). PF-06928215 displays no inhibitory potency against dsDNA-induced IFN-β expression in cellular cGAS assays, most likely due to limited cell-permeability and/or high levels of cellular ATP and GTP in the reporter cells employed.
| Targets |
In the cellular cGAS experiment assessing dsDNA-induced IFN-β production, PF-06928215 did not exhibit any action [1].
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| ln Vitro |
In the cellular cGAS experiment assessing dsDNA-induced IFN-β production, PF-06928215 did not exhibit any action [1].
PF-06928215 bound to cGAS with high affinity (KD = 200 nM) as measured by surface plasmon resonance (SPR). [1] PF-06928215 inhibited cGAS enzymatic activity in a fluorescence polarization (FP) assay with an IC₅₀ value of 4.9 μM. The assay measured inhibition of cGAMP production from ATP and GTP in the presence of dsDNA activator. [1] PF-06928215 displayed longer off-rate kinetics compared to earlier analogs in the series, as observed in SPR sensorgrams. [1] PF-06928215 showed no activity in a cellular assay measuring dsDNA-induced IFN-beta expression via a luciferase reporter in THP-1 Dual cells, even at high concentrations. This lack of activity was not due to cytotoxicity. [1] X-ray crystallography revealed that PF-06928215 binds to the active site of cGAS. Its alkyl chain interacts with a hydrophobic pocket formed between Tyr436 and His437, while its polar head groups interact with polar residues Arg376 and Lys362. The carboxylate group of PF-06928215 interacts with Lys362, mimicking an interaction observed in a linear reaction intermediate of cGAS catalysis. [1] |
| Enzyme Assay |
cGAS Activity Mass Spectrometry Assay: cGAS activity was measured in a buffer containing ATP, GTP, and a dsDNA activator (ISD DNA). The reaction was incubated, stopped with EDTA, and the product (cGAMP) was quantified using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Specific multiple reaction monitoring transitions were used to detect cGAMP, ATP, and GTP. This assay was used to characterize enzyme kinetics and for initial compound testing. [1]
cGAS Fluorescence Polarization (FP) Assay: cGAS activity was measured in a buffer containing ATP, GTP, and dsDNA activator. After incubation, the reaction was stopped with EDTA. Cy5-labeled cGAMP and a high-affinity monoclonal antibody specific for cGAMP (mAb 80-2) were added. The binding of the antibody to the fluorescent cGAMP analog generated a fluorescence polarization signal inversely proportional to the amount of unlabeled cGAMP produced by cGAS. This assay was used for high-throughput screening and potency determination of inhibitors like PF-06928215. [1] |
| Cell Assay |
Cellular cGAS/IFN-β Reporter Assay: THP-1 Dual cells (which contain an IFN-β-responsive luciferase reporter) were pretreated with various concentrations of PF-06928215 or control compounds for 1 hour. Cells were then stimulated with salmon sperm DNA for 12 hours to activate the cGAS-STING pathway. The cell culture media was collected, and the induction of IFN-β was assessed by measuring the luciferase signal. Cell viability was assessed in parallel using a luminescent cell viability assay to rule out cytotoxicity as the cause of any observed inhibition. [1]
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| References | |
| Additional Infomation |
PF-06928215 is a high-affinity cGAS active site inhibitor developed from a lead compound fragment via structure-based drug design. [1]
Its discovery aims to address the potential therapeutic need for cGAS inhibition in autoimmune diseases such as systemic lupus erythematosus, which are associated with aberrant cGAS activation. [1] Developing a novel high-throughput fluorescent polarization detection method using a cGAMP-specific monoclonal antibody is key to the discovery and optimization of this series of compounds. [1] Despite its high biochemical affinity, PF-06928215 lacks cellular activity, suggesting a need to further improve its potency, passive penetration, and plasma protein binding to achieve cellular and in vivo therapeutic effects. [1] PF-06928215 is now marketed. [1] |
| Molecular Formula |
C20H20N4O4
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| Molecular Weight |
380.3972
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| Exact Mass |
380.148
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| CAS # |
2378173-15-8
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| PubChem CID |
137300622
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| Appearance |
White to off-white solid powder
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| Density |
1.5±0.1 g/cm3
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| Index of Refraction |
1.725
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| LogP |
3.19
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
28
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| Complexity |
779
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| Defined Atom Stereocenter Count |
2
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| SMILES |
C1CC[C@@H]([C@@H](C1)C(=O)O)NC(=O)C2=CNN3C2=NC(=CC3=O)C4=CC=CC=C4
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| InChi Key |
WGQGCLHXUPGUSZ-HIFRSBDPSA-N
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| InChi Code |
InChI=1S/C20H20N4O4/c25-17-10-16(12-6-2-1-3-7-12)22-18-14(11-21-24(17)18)19(26)23-15-9-5-4-8-13(15)20(27)28/h1-3,6-7,10-11,13,15,21H,4-5,8-9H2,(H,23,26)(H,27,28)/t13-,15+/m1/s1
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| Chemical Name |
(1R,2S)-2-[(7-oxo-5-phenyl-1H-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]cyclohexane-1-carboxylic acid
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| Synonyms |
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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 |
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| 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) |
DMSO : ~33.33 mg/mL (~87.62 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.43 mg/mL (3.76 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 14.3 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. Solubility in Formulation 2: ≥ 1.43 mg/mL (3.76 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 14.3 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. View More
Solubility in Formulation 3: ≥ 1.43 mg/mL (3.76 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.6288 mL | 13.1441 mL | 26.2881 mL | |
| 5 mM | 0.5258 mL | 2.6288 mL | 5.2576 mL | |
| 10 mM | 0.2629 mL | 1.3144 mL | 2.6288 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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