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VTX-27 (VTX27) is a novel, potent and selective protein kinase C θ (PKC θ) inhibitor with Ki values of 0.08 nM and 16 nM for PKC θ and PKC δ. Protein kinase C θ (PKCθ) has a central role in T cell activation and survival; however, the dependency of T cell responses to the inhibition of this enzyme appears to be dictated by the nature of the antigen and by the inflammatory environment.
| Targets |
Protein kinase C theta (PKCθ) (Ki = 0.08 nM), Protein kinase C delta (PKCδ) (Ki = 16 nM), Protein kinase C alpha (PKCα) (Ki = 356 nM), Other PKC isoforms: βI (Ki = 15 nM), βII (Ki = 393 nM), γ (Ki = 725 nM), ε (Ki = 1 nM), η (Ki = 3.5 nM), μ (Ki = 2420 nM), τ (Ki > 5000 nM), Other kinases in TCR pathway (Lck, Itk, Fyn, Lyn, Zap70, c-Raf, Erk-1): Ki > 1000 nM [1]
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| ln Vitro |
VTX-27 (compound 27) exhibits outstanding comprehensive properties. Other PKC family members are likewise selectively bound by VTX-27, particularly classical isoforms (>1000-fold, with the exception of PKCβI, 200-fold) and atypical isoforms (>10000-fold). As anticipated, it proved more difficult to achieve 200-fold selectivity over PKC delta for the more closely related new PKC family members [1].
VTX-27 is a potent and selective inhibitor of PKCθ enzyme with a Ki of 0.08 nM. It exhibits excellent selectivity over PKCδ (200-fold, Ki = 16 nM) and other PKC isoforms, particularly classical (e.g., PKCα, >4400-fold) and atypical isoforms (>10,000-fold). In a cell-based assay using anti-CD3/CD28 stimulated human peripheral blood mononuclear cells (PBMCs), VTX-27 inhibited IL-2 production with an IC₅₀ of 11 nM. In a more physiologically relevant human whole blood (WB) IL-2 assay, VTX-27 showed an IC₅₀ of 70 nM. It demonstrated no significant cross-reactivity against a panel of other proximal T-cell receptor (TCR) signaling kinases (e.g., Src, Syk, Tec, MAP kinases) at concentrations up to 1000 nM. [1] |
| ln Vivo |
VTX-27 exhibited ideal pharmacological characteristics, including a long half-life (4.7 hours), low clearance (7 mL min-1 kg-1), and good oral bioavailability (65%). Single doses of VTX-27 showed strong dose-dependent suppression of IL-2 production when given orally at 6.25, 12.5, 25, and 50 mg/kg (e.g., 25 mg/kg Cmax concentration 700 ng/mL) [1].
In a murine model of staphylococcal enterotoxin B (SEB)-induced IL-2 release, a single oral dose of VTX-27 (6.25, 12.5, 25, and 50 mg/kg) produced a potent and dose-dependent inhibition of plasma IL-2 levels. At 25 mg/kg (Cmax ~700 ng/mL), VTX-27 significantly inhibited IL-2 production. The positive control cyclosporin A (15 mg/kg) also showed significant inhibition. [1] |
| Enzyme Assay |
PKCθ and PKCδ Radiometric Assay: The assay measured the transfer of the gamma-phosphate of ATP to a biotinylated peptide substrate. Reactions contained recombinant enzyme, peptide substrate, [γ-³³P]ATP, lipids (phosphatidylserine and diacylglycerol), and test compound in DMSO. After 60 minutes, reactions were stopped with phosphoric acid and transferred to a phosphocellulose filter plate. Unincorporated ATP was washed away, and radioactivity was quantified by scintillation counting. Ki values were determined by fitting data to a competitive tight-binding inhibition model. PKCα and PKCζ Spectrophotometric Assay: This coupled enzyme assay monitored NADH consumption at 340 nm. Reactions contained full-length recombinant enzyme, lipid cofactors, substrate peptide, ATP, NADH, phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase. Serial compound dilutions were added in DMSO, and reactions were initiated with ATP. Initial rates were determined from absorbance change over 15 minutes. Ki values were calculated using a competitive tight-binding inhibition model. [1]
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| Cell Assay |
PBMC T Cell IL-2 Assay: Human PBMCs were isolated from blood, washed, and resuspended. Cells were added to 96-well plates pre-coated with anti-CD3 and anti-CD28 antibodies, along with serial dilutions of VTX-27 or DMSO vehicle. Plates were incubated for 24 hours. Supernatants were collected, and IL-2 levels were quantified using a bead-based ELISA. IC₅₀ values were determined by nonlinear regression analysis. Whole Blood (WB) IL-2 Assay: Human whole blood was dispensed into a 96-well plate. Serial dilutions of VTX-27 or vehicle were added and incubated for 1 hour with shaking. Phytohemagglutinin (PHA) was then added to stimulate T cells, and incubation continued for 6 hours. Plates were centrifuged to pellet cells, supernatants were collected, and IL-2 levels were measured using a standard ELISA. [1]
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| Animal Protocol |
SEB-Induced IL-2 Release Model in Mice: Female Balb/c mice were used. VTX-27 was formulated as a solution in 1% Tween 80 / 99% sodium citrate buffer (pH 3). Animals received a single oral dose (6.25 to 50 mg/kg, 10 mL/kg) of VTX-27, vehicle, or cyclosporin A (15 mg/kg) as a reference. One hour after compound administration, mice were injected intraperitoneally with SEB. Two hours post-SEB challenge, terminal blood samples were collected. Plasma was isolated and analyzed for IL-2 concentration using a sandwich enzyme immunoassay. Statistical analysis was performed using one-way ANOVA followed by Dunnett‘s post-test. [1]
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| ADME/Pharmacokinetics |
In pharmacokinetic studies of rats after intravenous (3 mg/kg) and oral (10 mg/kg) administration, VTX-27 exhibited low plasma clearance (CL = 7 mL min⁻¹ kg⁻¹), a long half-life (t₁/₂ = 4.7 h), and good oral bioavailability (F = 65%). [1]
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| References | |
| Additional Infomation |
VTX-27 is a novel, potent, and selective PKCθ inhibitor designed for the treatment of autoimmune diseases. It is designed based on a pyrazolo[3,4-b]pyridine skeleton. The compound achieves selectivity by exploiting the subtle structural differences between PKCθ and its closely related subtypes, such as PKCδ, in the hydrophobic cavity near the ATP binding site. This selectivity is intended to inhibit pathogenic T cell responses in autoimmune diseases without causing widespread immunosuppression, and therefore may have better safety compared to pan-PKC inhibitors. [1]
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| Molecular Formula |
C₂₀H₂₄CLFN₆O
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|---|---|
| Molecular Weight |
418.90
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| Exact Mass |
418.168
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| CAS # |
1321924-70-2
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| PubChem CID |
53354074
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| Appearance |
White to off-white solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
625.8±55.0 °C at 760 mmHg
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| Flash Point |
332.3±31.5 °C
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| Vapour Pressure |
0.0±1.9 mmHg at 25°C
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| Index of Refraction |
1.624
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| LogP |
2.48
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
29
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| Complexity |
573
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CC(C)[C@](C)([C@@H]1CN(CCN1)C2=C(C=C(C(=N2)C3=C4C=CC=NC4=NN3)F)Cl)O
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| InChi Key |
HXWARSZQGAFXJM-MGPUTAFESA-N
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| InChi Code |
InChI=1S/C20H24ClFN6O/c1-11(2)20(3,29)15-10-28(8-7-23-15)19-13(21)9-14(22)17(25-19)16-12-5-4-6-24-18(12)27-26-16/h4-6,9,11,15,23,29H,7-8,10H2,1-3H3,(H,24,26,27)/t15-,20+/m0/s1
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| Chemical Name |
(2R)-2-[(2S)-4-[3-chloro-5-fluoro-6-(2H-pyrazolo[3,4-b]pyridin-3-yl)pyridin-2-yl]piperazin-2-yl]-3-methylbutan-2-ol
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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) |
DMSO : ~125 mg/mL (~298.40 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.97 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 20.8 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: ≥ 2.08 mg/mL (4.97 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 20.8 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: ≥ 2.08 mg/mL (4.97 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 | 2.3872 mL | 11.9360 mL | 23.8720 mL | |
| 5 mM | 0.4774 mL | 2.3872 mL | 4.7744 mL | |
| 10 mM | 0.2387 mL | 1.1936 mL | 2.3872 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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