PKCι (atypical protein kinase C iota) [1].
CDK9/cyclin T1 complex - disruption of interaction observed [1].

Oncrasin-1 can selectively kill K-Ras mutant cancer cells and induce abnormal nuclear aggregation of PKCiota in sensitive cells but not in resistant cells. Oncrasin-1 treatment led to coaggregation of PKCiota and splicing factors into megaspliceosomes but had no obvious effects on the DNA repair molecule Rad51. Moreover, oncrasin-1 treatment suppressed the phosphorylation of the largest subunit of RNA polymerase II and the expression of intronless reporter genes in sensitive cells but not in resistant cells, suggesting that suppression of RNA transcription is a major effect of oncrasin-1 treatment. Studies with cultured cells or with recombinant proteins showed that oncrasin-1 can disrupt the interaction of PKCiota and cyclin-dependent protein kinase 9/cyclin T1 complex, which is known to phosphorylate the largest subunit of RNA polymerase II and is required for RNA transcription. Together, our results suggest that oncrasin-1 suppresses the function of RNA processing machinery and that PKCiota might be involved in the biological function of RNA processing complexes

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Oncrasin-1 treatment (10 μmol/L for T29/T29Kt1; 1 μmol/L for H460) induced aggregation of PKCι into large nuclear foci in sensitive T29Kt1 cells after 12 h, but not in resistant cells [1].
Oncrasin-1 caused aggregation of RNA splicing factors SC35 and ASF/SF2 into megaspliceosomes in T29Kt1 cells, with colocalization with PKCι observed by immunofluorescent staining after 12 h treatment at 10 μmol/L [1].
Oncrasin-1 had no obvious effect on the DNA repair protein Rad51 in T29Kt1 cells after 12 h [1].
Oncrasin-1 treatment (10 μmol/L for T29/T29Kt1; 1 μmol/L for H460) dramatically suppressed phosphorylation of the largest subunit of RNA polymerase II (detected by H5 antibody) and phosphorylation of SR proteins in sensitive T29Kt1 and H460 cells at 12 h, but not in T29 (normal) or in resistant H1299 (N-Ras mutant) and H322 (wild-type Ras) cells [1].
Time-course study in H460 cells: Oncrasin-1 (1 μmol/L) reduced phosphorylation of RNA polymerase II and SR proteins starting at 8 h, with more striking effects at 12 and 24 h [1].
Oncrasin-1 (1 μmol/L for H460, 10 μmol/L for T29/T29Kt1) markedly inhibited luciferase expression from intronless reporter plasmids (pCMV-Luc and pRL-TK-Luc) in H460 and T29Kt1 cells, but had no or mild effect in T29 cells; inhibition was stronger than that of 100 μmol/L DRB, measured at 4, 8, and 12 h post-treatment [1].
Immunoprecipitation in H460 cells showed that PKCι co-precipitated with cyclin T1 in DMSO control; after Oncrasin-1 (1 μmol/L, 12 h) treatment, PKCι was not detected in the CDK9/cyclin T1 complex [1].
In vitro binding assay: recombinant PKCι and CDK9/cyclin T1 were mixed with or without 1 μmol/L Oncrasin-1; anti-PKCι antibody precipitated cyclin T1 in the absence but not in the presence of Oncrasin-1, indicating disruption of the interaction [1].
Oncrasin-1 at high concentrations (dose-dependent, exact IC50 not provided) inhibited the kinase activity of recombinant CDK6 and CDK9, and to a moderate extent also CDK2 and CDK7, as shown by in vitro kinase assays (Supplementary Fig. S3) [1].

The kinase reactions (20 µL) were performed at 30°C for 15 min. The solution contained 1X reaction buffer, 5 mM MgCl2, 50 µM cold ATP, 1 µL γ[P32]-ATP (3000 Ci/mmol), 50 ng individual kinase, and their corresponding substrates. 500mM CDK7/9 tide was used as the substrate for CDK9/cyclin T1, CDK7/cyclin H/MAT1, while 0.1mg/ml Histone H1 was used for CDK2/cyclinA, CDK6/cyclinD3 kinase assay. The mix was incubated with different doses of oncrasin-1. Free γ[P32]-ATP was washed away through P81 phosphocellulose filters. The samples were read under a scintillation counter.

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Kinase activity assay: Kinase reactions (20 μL) were performed at 30°C for 15 min. The reaction mixture contained 1× reaction buffer, 5 mmol/L MgCl2, 50 μmol/L cold ATP, 1 μL γ[P32]ATP (3000 Ci/mmol), 50 ng of each recombinant kinase (CDK2/cyclin A, CDK6/cyclin D3, CDK7/cyclin H/MAT1, or CDK9/cyclin T1), and corresponding substrates (500 mmol/L CDK7/9 tide for CDK7/9; 0.1 mg/mL histone H1 for CDK2/6). Different doses of Oncrasin-1 were added to the mixture. Free γ[P32]ATP was removed by washing through P81 phosphocellulose filters, and samples were counted in a scintillation counter. Oncrasin-1 inhibited CDK6 and CDK9 in a dose-dependent manner (exact IC50 not provided) [1].
In vitro binding assay: Recombinant PKCι and CDK9/cyclin T1 complex were mixed with or without 1 μmol/L Oncrasin-1 at 4°C for 2 h with gentle mixing. The mixture was then precipitated with anti-PKCι antibody (normal rabbit IgG as control). Precipitates were analyzed by Western blot for cyclin T1. The presence of Oncrasin-1 dramatically suppressed co-immunoprecipitation of PKCι and cyclin T1 [1].

Cells (3×104/well) were seeded in 24-well plates overnight. They were then grown in serum-free medium for 12 h, followed by transfection with 250 ng of different luciferase reporter plasmids. FuGENE 6 was used for plasmid transfection. The cells were kept in normal medium for another 24 h and then treated with 1 µM oncrasin-1 for different time periods. Cells were harvested for the luciferase activity assay, which was performed using the luciferase assay system (Promega Life Science) as instructed by the manufacturer. Cells transfected with pCDNA3.1 were used as the control.[1]

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Cell lines: Human non-small cell lung carcinoma H460 (K-Ras mutant), T29 (immortalized normal ovarian epithelial), T29Kt1 (K-Ras transformed), H1299 (N-Ras mutant), H322 (wild-type Ras). Cells were cultured in RPMI 1640 or DMEM with 10% FBS, penicillin/streptomycin at 37°C in 5% CO2 [1].
Immunofluorescent staining: Cells seeded at 1×10^5 per well in 6-well plates with gelatin-treated coverslides. After treatment with Oncrasin-1 (10 μmol/L for T29/T29Kt1; 1 μmol/L for H460) or DMSO or 10-Gy γ irradiation for 12 h, cells were fixed with 2% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 20 min, blocked with 5% normal goat serum for 1 h. Slides incubated with primary antibodies (anti-PKCι, anti-SC35, anti-ASF/SF2, anti-Rad51), then with FITC- or rhodamine-linked secondary antibodies. Mounted with Prolong Gold antifade containing DAPI. Examined under fluorescence or confocal microscope [1].
Western blot: Cell lysates prepared in RIPA buffer (50 mmol/L Tris-HCl pH 8, 150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS), passed through 21-gauge needles 10 times, centrifuged at 10,000 rpm for 10 min. Supernatant used for SDS-PAGE and Western blot with antibodies against phosphorylated RNA polymerase II (H5), phosphorylated SR proteins, PKCι, cyclin T1, β-actin, etc. [1].
Immunoprecipitation: Cell lysates incubated with primary antibodies overnight under mild rotation, then 25 μL IgA/G beads added and shaken for ~1 h. Beads washed, diluted with loading buffer, heated at 95°C for 10 min, then subjected to SDS-PAGE and Western blot [1].
Luciferase assay: Cells (3×10^4/well) seeded in 24-well plates, grown in serum-free medium for 12 h, then transfected with 250 ng pCMV-Luc or pRL-TK-Luc plasmids using FuGENE 6. After 24 h, treated with Oncrasin-1 (1 μmol/L for H460; 10 μmol/L for T29/T29Kt1), DMSO, or 100 μmol/L DRB for indicated times (4, 8, 12 h). Cells harvested for luciferase activity assay using luciferase assay system; values normalized to DMSO control [1].
Flow cytometry: Not described in this paper for cell assay (only mentioned in results but not in methods? Actually flow cytometry was used to assess differentiation rate in a different context? No, that was for SJA710-6, not for Oncrasin-1. For Oncrasin-1, no flow cytometry details are provided in this paper. So not include.)

Mol Cancer Ther. 2009 Feb;8(2):441-8.

Oncrasin-1 is an indole compound with the structure 1H-indole, substituted at positions 1 and 3 with 4-chlorobenzyl and formyl groups, respectively. It is an anticancer drug active against lung cancer cells carrying K-Ras mutations. It exhibits dual effects of inducing apoptosis and antitumor activity. Oncrasin-1 belongs to the indole, monochlorobenzene, and aromatic formaldehyde classes of compounds.

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Oncrasin-1 was originally identified through a synthetic lethality screen in K-Ras mutant cancer cells. The compound selectively kills K-Ras mutant cancer cells but has little effect on normal isogenic cells. Its anti-tumor activity is not affected by Raf inhibitor Bay 43-9006, MEK inhibitor U0126, PI3K inhibitor LY294002, or AKT inhibitor X. The compound induces aggregation of PKCι and splicing factors into megaspliceosomes, suppresses RNA polymerase II CTD phosphorylation, and disrupts the interaction between PKCι and the CDK9/cyclin T1 complex. This leads to inhibition of RNA transcription and ultimately cell death. The University of Texas M.D. Anderson Cancer Center filed a patent for Oncrasin-1; W. Guo, S. Wu, J. Liu, and B. Fang are the inventors [1].

C16H12CLNO

269.7256

269.061

75629-57-1

872445

Light yellow to khaki solid powder

1.21g/cm3

465.5ºC at 760 mmHg

117 °C

235.3ºC

1.62

4.155

0

1

3

19

314

0

ZDRQMXCSSAPUMM-UHFFFAOYSA-N

InChI=1S/C16H12ClNO/c17-14-7-5-12(6-8-14)9-18-10-13(11-19)15-3-1-2-4-16(15)18/h1-8,10-11H,9H2

1-(4-Chlorobenzyl)-1H-indole-3-carbaldehyde

Oncrasin-1; Oncrasin 1; Oncrasin1

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 (~370.74 mM)

Solubility in Formulation 1: 2.5 mg/mL (9.27 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (9.27 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 (9.27 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear 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 corn oil and mix evenly.

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