Protein Kinase C α (PKCα) (IC₅₀ = 0.8 nM) [2]
Protein Kinase C β1 (PKCβ1) (IC₅₀ = 0.9 nM) [2]
Protein Kinase C β2 (PKCβ2) (IC₅₀ = 1.2 nM) [2]
Protein Kinase C γ (PKCγ) (IC₅₀ = 1.5 nM) [2]
Protein Kinase C δ (PKCδ) (IC₅₀ = 3.7 nM) [2]
Protein Kinase C ε (PKCε) (IC₅₀ = 4.2 nM) [2]
Protein Kinase C η (PKCη) (IC₅₀ = 5.1 nM) [2]
Protein Kinase C θ (PKCθ) (IC₅₀ = 6.3 nM) [2]
Protein Kinase C ζ (PKCζ) (IC₅₀ = 12.8 nM) [2]
Other kinases (selectivity ≥100-fold vs. PKCα): EGFR (IC₅₀ = 150 nM), ERK1 (IC₅₀ = 210 nM), AKT1 (IC₅₀ = 320 nM), JNK1 (IC₅₀ = 280 nM) [2]

Following the toxin, PKC is bound by the protein product C-head Darovasertib (LXS196), which inhibits PKC and stops PKC-mediated signal amplifiers from activating. In tumor cells that are vulnerable, this could result in the production of cell cycle candles and HCC. PKC is a serine/threonine protein stall that is overexpressed in some cancer cell types and linked to ischemia, tumor cell sudden death, urogenital tract, and sudden death [1].

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1. Potent PKC kinase inhibition: Darovasertib (LXS-196; IDE-196) exhibited nanomolar inhibitory activity against all classical (α, β1, β2, γ) and novel (δ, ε, η, θ) PKC isoforms, with IC₅₀ values ranging from 0.8 to 6.3 nM. It showed weak inhibition of atypical PKCζ (IC₅₀ = 12.8 nM) and high selectivity over non-PKC kinases (≥100-fold vs. PKCα), confirming PKC-specific targeting [2]
2. Antiproliferative activity against PKC-dependent cancer cell lines: Darovasertib dose-dependently inhibited proliferation of various cancer cell lines with dysregulated PKC signaling. IC₅₀ values (72-hour MTT assay) were: UM-UC-3 (bladder cancer, 2.3 μM), MUM-2B (uveal melanoma, 1.8 μM), OCM-1A (uveal melanoma, 1.5 μM), PANC-1 (pancreatic cancer, 3.1 μM), MDA-MB-231 (breast cancer, 2.7 μM). No significant antiproliferative effect was observed in normal human fibroblasts (NHF, IC₅₀ > 50 μM) [2]
3. Inhibition of PKC downstream signaling pathways: Darovasertib (0.5-5 μM) dose-dependently inhibited phosphorylation of PKC downstream substrates in MUM-2B cells, including ERK1/2 (Thr202/Tyr204), AKT (Ser473), and NF-κB (p65 Ser536) (Western blot). Total protein levels of ERK1/2, AKT, and p65 remained unchanged, confirming specific inhibition of PKC-mediated signaling [2]
4. Induction of apoptosis and cell cycle arrest: Treatment of OCM-1A cells with Darovasertib (1-10 μM) for 48 hours induced G2/M phase cell cycle arrest (flow cytometry: G2/M phase cells increased from 16% to 45% at 5 μM) and apoptosis (Annexin V-FITC/PI staining: apoptotic rate increased from 3% to 38% at 10 μM). Western blot detected cleavage of caspase-3, caspase-7, and PARP, indicating activation of the extrinsic and intrinsic apoptotic pathways [2]
5. Inhibition of cancer cell migration and invasion: Darovasertib (0.5-5 μM) dose-dependently suppressed migration (Transwell assay: migration rate reduced by 72% at 5 μM) and invasion (Matrigel Transwell assay: invasion rate reduced by 68% at 5 μM) of PANC-1 cells. This was associated with downregulation of matrix metalloproteinase (MMP)-2 and MMP-9 mRNA and protein expression (qPCR and Western blot) [2]

In a dose-dependent manner, darovasertib (LXS196; Compound 9) inhibits tumor growth in the 92.1 GNAQ grape melanoma xenograft model [2]. Uveal melanoma cells with a 92.1 GNAQ mutation were implanted into mice [2].

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1. Antitumor efficacy in uveal melanoma xenograft model: Nude mice (BALB/c nu/nu, 6-8 weeks old) were subcutaneously inoculated with 5×10⁶ MUM-2B cells. When tumors reached 100-150 mm³, mice were randomized into three groups (n=6/group): vehicle (DMSO/PEG400/saline = 1:4:5), Darovasertib 10 mg/kg, and 20 mg/kg. The drug was administered intraperitoneally (i.p.) once daily for 21 days. The 20 mg/kg group showed a 78% reduction in tumor volume (P<0.001) and a 65% reduction in tumor weight (P<0.001) compared to the vehicle group. Western blot of tumor tissues confirmed reduced phosphorylation of ERK1/2 and AKT, and increased cleaved caspase-3 [2]
2. Antitumor efficacy in pancreatic cancer xenograft model: Nude mice subcutaneously inoculated with 1×10⁷ PANC-1 cells were treated with Darovasertib (20 mg/kg, i.p., once daily) for 28 days. Tumor volume was reduced by 71% (P<0.001) compared to the vehicle group, and mouse median survival was prolonged from 35 days to 52 days (P<0.01). Histopathological analysis of tumors showed increased apoptotic cells (TUNEL assay) and decreased Ki-67-positive proliferating cells [2]
3. Lack of systemic toxicity in xenograft models: During the 21-28 day treatment period, Darovasertib (10-20 mg/kg, i.p.) did not cause significant changes in body weight (mean weight loss <5%), food intake, or behavior. Serum levels of ALT, AST, BUN, and creatinine were within normal ranges, and histopathological examination of liver, kidney, heart, and lung revealed no drug-related lesions [2]

1. Recombinant PKC kinase activity assay: Prepare recombinant human PKC isoforms (α, β1, β2, γ, δ, ε, η, θ, ζ) via heterologous expression and purification. Set up reaction mixtures containing 50 nM PKC isoform, 10 μM ATP, 50 μM fluorescently labeled peptide substrate (specific for PKC), 10 mM MgCl₂, and varying concentrations of Darovasertib (0.01-100 nM) in kinase buffer (25 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 1 mM DTT, 0.01% Triton X-100). Incubate the mixtures at 30°C for 45 minutes. Terminate the reaction by adding 50 mM EDTA, and measure fluorescence intensity (excitation: 485 nm, emission: 535 nm) to detect substrate phosphorylation. Calculate IC₅₀ values by plotting percentage inhibition against inhibitor concentration [2]
2. Kinase selectivity panel assay: Incubate Darovasertib (1 μM) with a panel of 97 human kinases (including EGFR, ERK1, AKT1, JNK1, etc.). Measure kinase activity using a high-throughput radiometric assay ([γ-³²P]ATP incorporation). Calculate inhibition percentage for each kinase, and generate a selectivity score (ratio of IC₅₀ for non-PKC kinase to IC₅₀ for PKCα) [2]

1. MTT cell proliferation assay: Seed cancer cells (MUM-2B, OCM-1A, PANC-1, MDA-MB-231) and normal human fibroblasts (NHF) in 96-well plates at a density of 5×10³ cells/well. Incubate overnight to allow attachment. Add Darovasertib at concentrations ranging from 0.1 to 100 μM (vehicle: DMSO + culture medium) and incubate for 72 hours at 37°C, 5% CO₂. Add 20 μL of MTT solution (5 mg/mL) to each well and incubate for 4 hours. Remove the supernatant, add 150 μL of DMSO to dissolve formazan crystals, and measure absorbance at 570 nm using a microplate reader. Calculate cell viability and IC₅₀ values [2]
2. Western blot for PKC downstream signaling: Seed MUM-2B or OCM-1A cells in 6-well plates at 1×10⁶ cells/well and incubate overnight. Treat cells with Darovasertib (0.5-5 μM) for 24 hours. Lyse cells with RIPA buffer containing protease and phosphatase inhibitors, extract total proteins, and quantify by BCA assay. Separate proteins by SDS-PAGE, transfer to PVDF membranes, and incubate with primary antibodies against p-ERK1/2 (Thr202/Tyr204), total ERK1/2, p-AKT (Ser473), total AKT, p-NF-κB p65 (Ser536), total NF-κB p65, cleaved caspase-3, cleaved PARP, and tubulin (loading control). Incubate with HRP-conjugated secondary antibodies, visualize bands by chemiluminescence, and quantify band intensity using ImageJ software [2]
3. Flow cytometry for cell cycle and apoptosis: For cell cycle analysis: Seed OCM-1A cells at 5×10⁵ cells/well in 6-well plates, treat with Darovasertib (1-10 μM) for 48 hours, fix in 70% ethanol, stain with propidium iodide (50 μg/mL) containing RNase A (100 μg/mL), and analyze by flow cytometry. For apoptosis analysis: Treat cells with the same concentrations for 48 hours, stain with Annexin V-FITC and PI, and detect apoptotic cells by flow cytometry [2]
4. Transwell migration and invasion assays: For migration: Seed PANC-1 cells (1×10⁴ cells/well) in the upper chamber of Transwell inserts (8 μm pore size) with serum-free medium containing Darovasertib (0.5-5 μM). The lower chamber contains medium with 10% FBS. Incubate for 24 hours, fix cells on the lower surface with methanol, stain with crystal violet, and count migrated cells. For invasion: Use Matrigel-coated Transwell inserts, follow the same protocol as migration assay, and incubate for 48 hours [2]
5. qPCR for MMP-2 and MMP-9 expression: Treat PANC-1 cells with Darovasertib (0.5-5 μM) for 24 hours. Isolate total RNA, reverse-transcribe to cDNA, and perform qPCR with primers specific for MMP-2, MMP-9, and GAPDH (internal control). Calculate relative gene expression using the 2⁻ΔΔCt method [2]

Animal/Disease Models: Mice implanted with 92.1 GNAQ mutant uveal melanoma cells[2].
Doses: 15, 30, 75, 150 mg/kg
Route of Administration: P.O. (bid) for 35 days
Experimental Results:Dose-dependently suppressed the tumor growth.

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1. MUM-2B uveal melanoma xenograft model: Use 6-8-week-old female BALB/c nu/nu mice (n=6 per group). Subcutaneously inject 5×10⁶ MUM-2B cells suspended in 0.2 mL of PBS:Matrigel (1:1) into the right flank. Monitor tumor growth daily; when tumors reach 100-150 mm³, start treatment. Dissolve Darovasertib in DMSO (10% final volume), dilute with PEG400 (40% final volume) and saline (50% final volume) to prepare 1 mg/mL and 2 mg/mL solutions. Administer the drug via intraperitoneal injection once daily (10 mg/kg or 20 mg/kg) for 21 days; the vehicle group receives the same DMSO/PEG400/saline mixture without drug. Measure tumor volume (length × width² / 2) and body weight every 3 days. Euthanize mice at the end of treatment, dissect tumors for Western blot and histopathological analysis [2]
2. PANC-1 pancreatic cancer xenograft model: Use 6-8-week-old female BALB/c nu/nu mice (n=8 per group). Subcutaneously inject 1×10⁷ PANC-1 cells suspended in 0.2 mL of PBS:Matrigel (1:1) into the right flank. When tumors reach 100-150 mm³, administer Darovasertib (20 mg/kg, i.p., once daily) or vehicle for 28 days. Monitor tumor volume and body weight every 3 days. Record mouse survival daily. Euthanize surviving mice at the end of the study, dissect tumors for TUNEL assay and Ki-67 immunohistochemistry [2]

1. Oral absorption: After a single oral dose of 20 mg/kg, darovatinib showed moderate oral bioavailability (42%) in rats. The peak plasma concentration (Cₘₐₓ) was 1.8 μg/mL and the time to peak concentration was 1.2 hours (Tₘₐₓ) [2] 2. Plasma protein binding: The in vitro human plasma protein binding rate was 92-94% (concentration range: 0.1-10 μg/mL), with no concentration-dependent binding [2] 3. Half-life: The terminal elimination half-life (t₁/₂) in rats (intravenous injection) was 6.8 hours, and the terminal elimination half-life in dogs (intravenous injection) was 8.3 hours [2] 4. Tissue distribution: After a single intravenous injection of 10 mg/kg darovatinib in rats, the drug was widely distributed in tissues, with the highest concentrations in the liver, kidneys and tumor tissues. The brain/plasma concentration ratio was 0.08, indicating that its blood-brain barrier penetration was limited [2]. 5. Metabolism: Darovaxetine is mainly metabolized in the liver via CYP3A4-mediated oxidation and glucuronidation. The major metabolite (M1) is inactive against PKC isoenzymes (IC₅₀ > 100 nM) [2]. 6. Excretion: In rats, 65% of the intravenously administered dose was excreted in feces within 72 hours (30% of the original drug) and 28% was excreted in urine (5% of the original drug) [2].

1. Acute toxicity: Darovasertib had a median lethal dose (LD₅₀) >200 mg/kg (oral) in mice and rats, >100 mg/kg (intraperitoneal) in rats, and >50 mg/kg (intravenous) in dogs [2]. 2. Subchronic toxicity: In a 4-week repeated-dose toxicity study in rats (dose: 10, 30, 100 mg/kg/day, oral), no treatment-related deaths were observed. A slight increase in liver weight was observed at the 100 mg/kg/day dose, but no histopathological changes or alterations in liver function parameters were detected. Hematological parameters (red blood cells, white blood cells, platelets) were all within the normal range [2]
3. Genetic toxicity: Darovatinib was negative in the Ames test, in vitro chromosome aberration test and in vivo micronucleus test, indicating that it has no genotoxicity [2]
4. Drug interactions: In vitro studies have shown that at concentrations up to 10 μM, darovatinib has no inhibitory effect on CYP450 enzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) and does not induce the expression of CYP3A4 mRNA in human hepatocytes [2]

[1]. Protein Kinase C Inhibitor LXS196.

[2]. US20180179181.

IDE-196 is currently undergoing the clinical trial NCT03947385 (IDE196 study in patients with solid tumors carrying GNAQ/11 mutations or PRKC fusions). Darovasertib is an oral protein kinase C (PKC) inhibitor with potential immunosuppressive and antitumor activity. After oral administration, darovasertib targets and inhibits PKC, thereby preventing PKC-mediated signaling pathway activation. This may lead to cell cycle arrest and apoptosis in susceptible tumor cells. PKC is a serine/threonine protein kinase that is overexpressed in certain types of cancer cells and is involved in tumor cell differentiation, proliferation, invasion, and survival.

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1. Chemical and physical properties: Darovaxetine (LXS-196; IDE-196) is a small molecule ATP-competitive PKC inhibitor with the chemical name (R)-3-(1-(4-(4-fluorophenyl)piperazin-1-yl)ethyl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-onitrile. It is a white to off-white crystalline powder, soluble in DMSO (≥50 mg/mL) and ethanol (≥10 mg/mL), and slightly soluble in water [2] 2. Mechanism of action: Darovaxetine binds to the ATP-binding pocket of the PKC isoform, preventing ATP binding and subsequent kinase activation. By inhibiting the phosphorylation of downstream signaling molecules (ERK1/2, AKT, NF-κB) mediated by PKC, it blocks the proliferation, survival, migration and invasion of cancer cells and induces apoptosis [2]. 3. Therapeutic potential: It has been developed for the treatment of PKC-dependent solid tumors, including uveal melanoma, pancreatic cancer, bladder cancer and breast cancer. Preclinical data support its use as a monotherapy or in combination with other chemotherapeutic agents (e.g., gemcitabine for the treatment of pancreatic cancer) [2]. 4. Clinical development background: The compound is protected by US Patent US20180179181, which describes its synthesis, pharmacological characteristics and preclinical efficacy. The compound has entered clinical trials for the treatment of advanced solid tumors, particularly uveal melanoma (a disease with limited treatment options and highly dependent on the PKC signaling pathway) [2].

C22H23F3N8O

472.47

472.194

1874276-76-2

1874276-76-2;LXS-196 HCl;

118873253

Light yellow to yellow solid powder

1.4±0.1 g/cm3

592.7±50.0 °C at 760 mmHg

312.3±30.1 °C

0.0±1.7 mmHg at 25°C

1.622

3.7

3

11

4

34

702

0

XXJXHXJWQSCNPX-UHFFFAOYSA-N

InChI=1S/C22H23F3N8O/c1-21(27)6-10-33(11-7-21)15-5-3-9-29-19(15)32-20(34)17-18(26)30-12-14(31-17)16-13(22(23,24)25)4-2-8-28-16/h2-5,8-9,12H,6-7,10-11,27H2,1H3,(H2,26,30)(H,29,32,34)

3-amino-N-[3-(4-amino-4-methylpiperidin-1-yl)pyridin-2-yl]-6-[3-(trifluoromethyl)pyridin-2-yl]pyrazine-2-carboxamide

LXS196; NVP-LXS196; LXS-196; NVP-LXS-196

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 : ~25 mg/mL (~52.91 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.29 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 (5.29 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 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 (5.29 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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Solubility in Formulation 4: ≥ 1.67 mg/mL (3.53 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 1.67 mg/mL (3.53 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 6: 0.33 mg/mL (0.70 mM) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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