Gli(IC50= 5 μM)
GLI-mediated transcription (downstream of Smoothened and Sufu in the Hedgehog signaling pathway). IC₅₀ ≈ 5 µM in cellular assays using SAG-treated Shh-L2 cells.[1]

Hh signaling is inhibited downstream by GANT58. Indeed, GANT58 inhibits Hh signaling by blocking Smo and Sufu. Because transcription induced by GLI1 with a mutated nuclear export signal is still blocked, GANT58 primarily functions at the nuclear level. GANT58 successfully inhibits the growth of tumor cells in vitro in a manner that is dependent on GLI. This is achieved by using human prostate cancer cells that have downstream activation of the Hh pathway to block cell growth[1].
It has been demonstrated that GANT58 (NSC75503) prevents GLI1 (as well as the other GLI species) from activating transcription. It has been demonstrated that GANT58 inhibits GLI transactivation[2].

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GANT58 was identified from a cell-based screen as a small-molecule antagonist of GLI-mediated transcription. In transfected HEK293 cells, it inhibited both GLI1- and constitutively active ΔN-GLI2 (GLI2β)-mediated transcription in a dose-dependent manner.[1]
In a more physiological system using the murine NIH 3T3 Shh-L2 cell line (stably incorporating a Gli reporter), GANT58 inhibited SAG-induced Hh signaling with an IC₅₀ of approximately 5 µM, comparable to the Smo inhibitor cyclopamine. The compound did not directly inhibit the reporter enzyme.[1]
In Ptch1⁻/⁻ mouse embryonic fibroblasts (MEFs), which exhibit constitutive Hh pathway activation, treatment with 10 µM GANT58 significantly reduced mRNA levels of the endogenous Hh target gene Gli1, indicating strong pathway inhibition. Similar inhibition was observed in assays measuring endogenous Ptch1 expression and SAG-induced alkaline phosphatase expression in C3H10T1/2 cells.[1]
In Sufu⁻/⁻ MEFs, where pathway activation occurs downstream of Smo, treatment with 10 µM GANT58 significantly reduced the high expression levels of Hh target genes Gli1 and Hip1 at both mRNA and protein levels. As expected, cyclopamine was ineffective in this model, confirming GANT58 acts downstream of Smo.[1]
GANT58 exhibited high selectivity for the Hh/Gli pathway, as treatment with up to 10 µM did not influence unrelated signaling pathways such as TNF/NF-κB, glucocorticoid receptor transactivation, or the Ras-Raf-MEK-MAPK cascade. It was also inactive in other reported screens for inhibitors of HGF/Met, C/EBPα, or HIF-1.[1]
GANT58 inhibited cellular transformation in NIH 3T3 cells transfected with a SHH expression plasmid, reducing colony formation in soft agar.[1]
In human tumor cell lines with elevated GLI1 levels (PANC1 pancreatic adenocarcinoma and 22Rv1 prostate carcinoma), treatment with 5 µM GANT58 for 48 hours reduced GLI1 and PTCH mRNA expression, consistent with GLI inhibition. In contrast, cyclopamine showed minimal effect, suggesting downstream pathway activation in these cells. Proliferation, as measured by BrdU incorporation, was inhibited by approximately 40-50% in these GLI1-high cell lines, but only marginally (0-10%) in low-GLI1 cell lines (HepG2, Jurkat). Cyclopamine had only marginal effects on proliferation.[1]
Mechanistically, GANT58 acts in the nucleus to block GLI function. It could inhibit a nuclear-accumulated GLI mutant as well as wild-type GLI. It did not affect primary cilia morphology/frequency (structures important for upstream Hh signaling). It did not induce phosphorylation of Creb, indicating its inhibition is not mediated via Protein Kinase A (PKA) activation.[1]

Tumors (median size ≈250 mm3) are created by injecting GLI1-positive 22Rv1 prostate cancer cells subcutaneously into nude mice. Every day, n = 4–5 injections of solvent alone, GANT61, GANT58, or cyclopamine at a dose of 50 mg/kg are given to naked mice. But after three days, the animals receiving cyclopamine showed signs of severe ulcerations at the injection sites. Consequently, the treatment plan was modified to include injections only once every two days.This protocol is also introduced for the GANTs so that all compounds can be compared, even though the mice treated with these compounds did not exhibit any toxicity. Every injection is made two to three centimeters away from the tumors. For all compounds, there is suppression of tumor cell growth during the course of an 18-day treatment period. The administration of GANT58 or cyclopamine inhibits the growth of additional xenografts and restricts the growth of tumors[1].

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In a human prostate cancer xenograft model using GLI1-positive 22Rv1 cells, established tumors (median size ~250 mm³) in nude mice were treated with GANT58 at 50 mg/kg via subcutaneous injection every second day for 18 days. Treatment inhibited additional xenograft growth and limited the increase in tumor size, but did not induce complete tumor regression (unlike GANT61). No adverse side effects such as weight loss, ulcerations, or signs of ill-being were observed during the treatment period.[1]
Analysis of tumors post-treatment showed a clear inhibition of cell proliferation (BrdU incorporation) compared to solvent control. Staining for cleaved caspase-3 indicated increased apoptosis in GANT58-treated samples compared to solvent or cyclopamine-treated tumors. Quantitative PCR analysis confirmed that GANT58 strongly reduced the expression of the Hh target gene PTCH in the xenografted cells.[1]

For the initial compound screen, HEK293 cells were transiently transfected with a GLI1 expression plasmid and a GLI-dependent firefly luciferase reporter plasmid (12xGliB-Luc). Cells were seeded in 96-well plates and treated with test compounds at a final concentration of 10 µM for 24 hours. Luciferase activity was measured using a dual-luciferase assay kit and normalized.[1]
For endogenous Hh signaling assays, Shh-L2 cells (NIH 3T3 with a stable Gli reporter) were treated with the Smo agonist SAG along with test compounds for 48 hours. Reporter activity (firefly luciferase) was measured and normalized to a control Renilla luciferase.[1]
For quantitative PCR analysis of endogenous target genes, cells (e.g., Ptch1⁻/⁻ MEFs, Sufu⁻/⁻ MEFs, PANC1, 22Rv1) were grown to confluency and treated with compounds (e.g., 10 µM) for 2-4 days. RNA was extracted, reverse transcribed, and mRNA levels of genes like Gli1, Hip1, or PTCH were quantified by real-time PCR and normalized to Gapdh or total protein.[1]
For BrdU incorporation proliferation assays, subconfluent cells were grown in medium with reduced serum (2.5% FBS) in the presence of 5 µM test compound or DMSO for 48 hours in 96-well plates. Cells were then labeled with BrdU for 2 hours, fixed, and BrdU incorporation was quantified using a luminometric cell proliferation ELISA kit.[1]

Female BALB/c nude mice were injected subcutaneously at the posterior flank with 5 x 10⁶ 22Rv1 human prostate cancer cells suspended in a 1:1 mixture of RPMI 1640 medium and Matrigel. Tumors were allowed to grow until they reached a median size of approximately 250 mm³ (5-6 days). Animals were then randomly divided into treatment groups (n=4-5 per group).
GANT58 was dissolved in a solvent consisting of corn oil and ethanol (4:1 ratio). The treatment regimen consisted of subcutaneous injections of 50 mg/kg GANT58 (or solvent control) every second day for a total of 18 days. Injections were administered several centimeters away from the tumor site. Tumor volumes were measured and calculated using the formula: length × width × 0.5 × (length + width). At the end of the treatment period, animals were given a BrdU pulse (50 mg/kg) 30 minutes before sacrifice, and tumors were excised for further analysis (histology, qPCR).[1]

In in vivo xenograft studies, mice were treated with 50 mg/kg GANT58 every other day for 18 days without adverse reactions such as weight loss, injection site ulceration, or general malaise. This contrasts sharply with cyclopamine treatment, which initially caused severe ulceration when administered daily. [1]
In vitro, treatment of several cell lines with concentrations sufficient to inhibit GLI (e.g., 5–10 µM) did not reveal significant toxicity or decreased cell viability, and therefore did not interfere with the assay results. [1]

[1]. Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists. Proc Natl Acad Sci U S A. 2007 May 15;104(20):8455-60.

[2]. GLI1 is a central mediator of EWS/FLI1 signaling in Ewing tumors. PLoS One. 2009 Oct 27;4(10):e7608.

4-(2,4,5-tripyridin-4-yl-3-thienyl)pyridine belongs to the pyridine class of compounds.

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GANT58 (NSC 75503) is a small molecule compound that was discovered through high-throughput screening of GLI-mediated transcription inhibitors, which is the final step in the Hedgehog (Hh) signaling pathway. Chemically, it has a thiophene core and four pyridine rings. [1] Its main significance lies in its ability to inhibit the Hh signaling pathway downstream of the transmembrane protein Smoothened (Smo) and the negative regulator Suppressor of Fused (Sufu), directly targeting GLI transcription factors. This makes it a potential tool for treating cancers associated with downstream activation of the Hh pathway, which are usually resistant to upstream Smo inhibitors such as cyclopamine. [1]
This compound exhibits high selectivity for the Hh/Gli pathway and demonstrates inhibitory and antiproliferative effects against Gli-dependent tumor cells both in vitro and in vivo. Furthermore, there is evidence that it can induce apoptosis in the tumor microenvironment in vivo. [1]

C24H16N4S

392.47564

392.109

C, 73.45; H, 4.11; N, 14.28; S, 8.17

64048-12-0

253078

Light yellow to yellow solid powder

1.3±0.1 g/cm3

414.1±40.0 °C at 760 mmHg

169.0±17.7 °C

0.0±0.9 mmHg at 25°C

1.659

3.12

0

5

4

29

449

0

C1(C2=C(C(C3=CC=NC=C3)=C(S2)C4=CC=NC=C4)C5=CC=NC=C5)=CC=NC=C1

USWLOKMMUTWFMD-UHFFFAOYSA-N

InChI=1S/C24H16N4S/c1-9-25-10-2-17(1)21-22(18-3-11-26-12-4-18)24(20-7-15-28-16-8-20)29-23(21)19-5-13-27-14-6-19/h1-16H

2,3,4,5-Tetra(4-pyridyl)thiophene

NSC75503; NSC-75503; NSC 75503; GANT 58; GANT-58.

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)

Ethanol : ~20 mg/mL (~50.96 mM)
DMSO : ~9.09 mg/mL (~23.16 mM)

Solubility in Formulation 1: 2 mg/mL (5.10 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear EtOH 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 mg/mL (5.10 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (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 20.0 mg/mL clear EtOH 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: ≥ 0.91 mg/mL (2.32 mM) 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 9.1 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 4: ≥ 0.91 mg/mL (2.32 mM) 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 9.1 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 5: ≥ 0.91 mg/mL (2.32 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 9.1 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 6: 10% EtOH+40% PEG300+5% Tween-80+45% Saline: 2 mg/mL (5.10 mM)

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Solubility in Formulation 7: 5 mg/mL (12.74 mM) in 45% PEG300 5% Tween-80 50% 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.)