Survivin; Op18
Survivin and Op18 expression are both potently and specifically inhibited by GDP366. Survivin and Op18 have lower mRNA and protein levels as a result of GDP366. Despite the fact that GDP366 significantly raises the levels of p53 and p21, this inhibitory effect is not reliant on their health. In vitro and in vivo (using a nude mouse model), GDP366 significantly slows the growth of tumor cells without quickly inducing apoptosis. Multiple kinds of cancer cell lines are induced to become polyploid by GDP366. By decreasing telomerase activity, GDP366 causes chromosomal instability to increase and accelerates cellular senescence[1].

Survivin and Op18 expression are both potently and specifically inhibited by GDP366. Survivin and Op18 have lower mRNA and protein levels as a result of GDP366. Despite the fact that GDP366 significantly raises the levels of p53 and p21, this inhibitory effect is not reliant on their health. In vitro and in vivo (using a nude mouse model), GDP366 significantly slows the growth of tumor cells without quickly inducing apoptosis. Multiple kinds of cancer cell lines are induced to become polyploid by GDP366. By decreasing telomerase activity, GDP366 causes chromosomal instability to increase and accelerates cellular senescence[1].

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GDP366 dose- and time-dependently decreased survivin and Op18 protein levels in HCT116 colon cancer cells, while increasing p53 and p21 levels, as shown by Western blot. [1]
Semi-quantitative RT-PCR showed that GDP366 decreased the mRNA levels of survivin and Op18 in a dose- and time-dependent manner in HCT116 and HeLa cells, without affecting HSP70 or HSP90 mRNA levels. [1]
The inhibitory effect of GDP366 on survivin and Op18 expression was independent of p53 and p21 status, as demonstrated in HCT116 p53-/- and p21-/- cells. [1]
GDP366 inhibited the clonogenic survival of HCT116 cells with an IC50 of approximately 1.0 µM in a colony formation assay. [1]
Sulforhodamine B protein biomass assays showed that GDP366 dose-dependently inhibited the growth of HCT116 p53+/+ cells (IC50 = 2.57 µM), HCT116 p53-/- cells (IC50 = 4.05 µM), HCT116 p21+/+ cells (IC50 = 0.95 µM), and HCT116 p21-/- cells (IC50 = 1.02 µM) after 72-hour treatment. [1]
GDP366 (2 µM) induced multinucleation, centrosome amplification, and the formation of micronuclei in KBM5 and HCT116 cells, indicative of mitotic catastrophe, as observed by phase-contrast, fluorescence (DAPI staining), and electron microscopy. [1]
Flow cytometry analysis of HCT116 cells treated with GDP366 (1-2 µM) for 24-72 hours showed a dose- and time-dependent increase in the proportion of polyploid (>4N DNA content) cells (up to 40-50%) and an eventual increase in sub-G1 population after longer exposure (72-96 h). This polyploidy induction was also independent of p53 and p21 status. [1]
GDP366 (2 µM) treatment for various durations induced chromosomal instability in HCT116 cells, evidenced by an increased proportion of cells with chromosomal aberrations (gaps and breaks). [1]
Western blot analysis showed that GDP366 treatment induced phosphorylation of histone H2AX (γH2AX), Chk1 (Ser345), and Chk2 (Thr68) in HCT116 cells in a dose- and time-dependent manner. [1]
GDP366 (2 µM) induced a senescence-like phenotype in HCT116 cells, as shown by increased activity of senescence-associated β-galactosidase (SA-β-gal) after 48-72 hours of treatment. [1]
A telomeric repeat amplification protocol (TRAP) assay showed that GDP366 (2 µM) treatment for 72 hours prominently inhibited telomerase activity in HCT116 cells. [1]
GDP366 (2 µM) treatment for 48 hours decreased the ratio of soluble (depolymerized) to polymerized tubulin in HCT116 cells, similar to the effect of the microtubule-stabilizing agent paclitaxel, indicating inhibition of tubulin polymerization. [1]

In a HCT116 xenograft nude mouse model, daily intraperitoneal (i.p.) administration of GDP366 at 50 mg/kg for 16 days (starting 5 days after tumor cell inoculation) significantly inhibited tumor growth compared to the vehicle control group. Tumor weights were also significantly lower in the treated group at the end of the study (day 21). [1]
The treatment was well-tolerated. No significant differences in body weight, motor activity, feeding behavior, or mortality were observed between the treated and control mice at this dosage. [1]

For Western blot analysis of protein levels, HCT116 or other cells were treated with GDP366 at indicated concentrations and durations. Whole cell lysates were prepared using RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts of protein were separated by SDS-PAGE, transferred to membranes, and probed with specific primary antibodies against survivin, Op18, p53, p21, γH2AX, phospho-Chk1, phospho-Chk2, etc., followed by HRP-conjugated secondary antibodies and detection. [1]
For semi-quantitative RT-PCR, total RNA was extracted from cells treated with GDP366 using Trizol reagent. cDNA was synthesized from 1 µg total RNA using random primers and a reverse transcription kit. PCR amplification was performed with gene-specific primers for survivin, Op18, HSP70, and GAPDH (as internal control). Products were separated on agarose gels and visualized with ethidium bromide. [1]
For the clonogenic survival assay, cells were seeded in six-well plates, exposed to GDP366 for 48 hours, then trypsinized and re-plated in fresh drug-free medium to grow for 10-14 days. Colonies were fixed with ethanol, stained with crystal violet, and those containing ≥50 cells were counted under a light microscope. [1]
For the sulforhodamine B (SRB) protein biomass assay, cells were treated with GDP366 in 96-well plates for 72 hours, fixed with cold trichloroacetic acid, washed, and stained with 0.4% SRB solution. Unbound dye was washed off with 1% acetic acid, and bound dye was solubilized with Tris base (pH 10.5). Absorbance was measured at 560 nm to estimate cell mass. [1]
For cell cycle analysis by flow cytometry, cells treated with GDP366 were collected, fixed in cold ethanol, washed, and stained with propidium iodide (PI) solution. DNA content was analyzed using a flow cytometer and appropriate software. [1]
For immunofluorescence staining, cells grown on coverslips were treated with GDP366, fixed with paraformaldehyde, permeabilized with Triton X-100/NP-40, blocked with serum, and incubated with primary antibodies (e.g., anti-lamin A/B, anti-α-tubulin, anti-γ-tubulin). After washing, cells were incubated with fluorescently labeled secondary antibodies, counterstained with DAPI for nuclei, mounted, and visualized by fluorescence or confocal microscopy. [1]
For senescence-associated β-galactosidase (SA-β-gal) staining, cells treated with GDP366 were fixed and incubated overnight at 37°C with a SA-β-gal staining solution according to the manufacturer's protocol. Blue-stained senescent cells were counted under a microscope. [1]
For the TRAP telomerase detection assay, telomerase was extracted from GDP366-treated cells. Telomerase extension was performed, followed by PCR amplification with specific primers. Products were separated on a non-denaturing polyacrylamide gel and visualized by silver staining. [1]
For measurement of soluble and polymerized tubulin, cells treated with GDP366 or control drugs (paclitaxel, vincristine) were lysed with a microtubule-stabilizing buffer. The lysate was centrifuged to separate the supernatant (soluble tubulin) from the pellet (polymerized tubulin). The pellet was further solubilized in SDS buffer. Both fractions were analyzed by Western blot using anti-tubulin antibody. [1]

Tumor Xenograft Model: Male nu/nu BALB/c mice (4-6 weeks old) were subcutaneously inoculated on the flank with 5 x 10^6 HCT116 cells. [1]
Treatment: Five days after inoculation, when tumors reached approximately 50 mm³, mice were randomized into control and treatment groups. GDP366 was dissolved in tissue-culture-grade DMSO and then diluted in tissue-culture medium (final DMSO concentration < 0.1% v/v). The treatment group received GDP366 at a dose of 50 mg/kg daily via intraperitoneal (i.p.) injection for 16 consecutive days. The control group received an equivalent volume of vehicle (DMSO in medium). [1]
Monitoring: Tumor dimensions were measured every other day with calipers, and tumor volume was calculated using the formula: (smallest diameter)² x (perpendicular diameter) x 0.4. Body weight, feeding behavior, and motor activity were monitored. Mice were euthanized on day 21 post-inoculation, tumors were excised and weighed. [1]

In the HCT116 xenograft study, daily intraperitoneal injection of 50 mg/kg GDP366 for 16 days was well tolerated. No significant toxicity was observed based on stable body weight, normal feeding behavior and exercise activity, and the absence of deaths. [1]

[1]. GDP366, a novel small molecule dual inhibitor of survivin and Op18, induces cell growth inhibition, cellular senescence and mitotic catastrophe in human cancer cells. Cancer Biol Ther. 2010 Apr 15;9(8):640-50.

GDP366 is a novel small molecule that, through compound library screening, was found to be a dual inhibitor of survivin and Op18. [1] Its mechanism of action involves selectively downregulating survivin and Op18 at the mRNA and protein levels, thereby inhibiting tubulin polymerization and inducing polyploidy, chromosomal instability, mitotic catastrophe, and cellular senescence. [1] This study indicates that GDP366 is a promising lead compound worthy of further translational research for cancer treatment. [1]

C20H17N5OS

375.44688

375.115

C, 63.98; H, 4.56; N, 18.65; O, 4.26; S, 8.54

501698-03-9

501698-03-9

6539985

Light yellow to yellow solid powder

3.8

3

5

3

27

513

0

DZSUJUOJJJCWGG-UHFFFAOYSA-N

InChI=1S/C20H17N5OS/c1-12-3-2-4-15(9-12)25-20(26)24-14-7-5-13(6-8-14)16-10-27-19-17(16)18(21)22-11-23-19/h2-11H,1H3,(H2,21,22,23)(H2,24,25,26)

1-[4-(4-aminothieno[2,3-d]pyrimidin-5-yl)phenyl]-3-(3-methylphenyl)urea

GDP 366; GDP366; GDP-366

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: ~62.5 mg/mL (~166.5 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.54 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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.

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