Ribosomal biogenesis regulator atypical kinase RIOK2 (Kd = 200 nmol/L) [1]
ERG (Ets-related gene) oncoprotein [1]

NSC139021 mostly suppresses the proliferation of ERG-positive cancer cell lines while having no effect on ERG-negative tumor cell lines, normal prostate cells, or endothelial cells. For sensitive cell lines, NSC139021's IC50 for inhibiting cell growth ranged from 30 nM to 400 nM. When combined, NSC139021 and enzalutamide had additive effects on the development of VCaP cells. NSC139021 directly binds the atypical kinase RIOK2, which regulates ribosome biogenesis, and causes ribosomal stress signals, according to kinase screening [1].

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ERGi-USU inhibited the growth of ERG-positive cancer cell lines VCaP (prostate), COLO320 (colon), KG-1 and MOLT-4 (leukemia) with IC50 values ranging from 30 to 400 nmol/L in an 8-day cell growth assay. [1]
ERGi-USU selectively inhibited ERG protein levels in ERG-positive cell lines (VCaP, COLO320, KG-1, MOLT-4). The IC50 for ERG protein inhibition in VCaP cells was 315 nmol/L. [1]
In contrast, ERG-negative cancer cell lines (LNCaP, LAPC4, MDA PCa2b) and normal cells (BPH-1, RWPE-1 prostate epithelial cells; HUVEC endothelial cells) showed minimal response to ERGi-USU, with IC50 values for growth inhibition exceeding 10 µmol/L. [1]
The combination of ERGi-USU (0.5 µmol/L) with enzalutamide (1 µmol/L) showed an additive effect, reducing VCaP cell growth by over 80%, compared to ~50% with ERGi-USU alone or ~20% with enzalutamide alone. [1]
ERGi-USU treatment (48 hours) induced ribosomal stress, as evidenced by decreased levels of RIOK2, phospho-S6RP, S6RP, and mTOR proteins in VCaP cells. [1]
Treatment with ERGi-USU also induced apoptosis, evidenced by cleavage of PARP-1, caspase-3, and caspase-7, and inhibited cell-cycle-associated proteins CDK4, cyclin D1, and cyclin D3 in VCaP cells. [1]
In VCaP cells, inhibition of ERG protein by ERGi-USU was evident at 18 hours, preceding the induction of cleaved PARP-1 at 24 hours. Co-treatment with the apoptosis inhibitor Z-VAD-FMK did not prevent ERG inhibition by ERGi-USU. [1]
ERGi-USU directly bound to and inhibited the RIOK2 protein. The IC50 values for RIOK2 protein inhibition were 220 nmol/L in VCaP cells and 360 nmol/L in COLO320 cells after 48 hours of treatment. [1]
A structure-activity relationship (SAR) study identified derivatives of ERGi-USU (ERGi-USU-2 and ERGi-USU-3) with similar potency in inhibiting ERG protein and cell growth. Replacing the naphthol ring with 5-alkyl-substituted phenols retained activity, while modifications to the thiazolyl ring or diazo linker abolished activity. [1]

Treatment with NSC139021 prevented ERG-positive VCaP tumor xenografts from growing without showing any signs of harm. Tumor growth was considerably (P<0.05, P<0.005) suppressed in the treatment groups on day 26, which corresponded to a 44% (100 mg/kg) and 65% (150 mg/kg) decrease in tumor burden. At the 100 mg/kg and 150 mg/kg dosages, no notable toxicities were noted, such as weight loss, somnolence, diarrhea, lack of appetite, respiratory distress, or overall drug-related toxicity. [1].

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In male nude mice bearing ERG-positive VCaP tumor xenografts, intraperitoneal administration of ERGi-USU at 100 mg/kg and 150 mg/kg (three times per week) significantly inhibited tumor growth compared to the vehicle control group. At day 26, tumor volume was reduced by 44% (100 mg/kg) and 65% (150 mg/kg). [1]
No apparent toxicity, including weight loss, lethargy, diarrhea, loss of appetite, respiratory distress, or gross damage to major organs and vasculature, was observed in mice treated with ERGi-USU. Localized inflammation at the injection site was rarely observed at the 150 mg/kg dose. [1]

A tryptophan fluorescence quenching assay was performed to confirm direct binding of ERGi-USU to human RIOK2 (HsRIOK2). Purified HsRIOK2 protein was prepared in a buffer containing Tris, NaCl, glycerol, and MgCl2. The intrinsic tryptophan fluorescence of the protein was measured after excitation at 295 nm. Emission spectra were collected for buffer blank, protein alone, and protein with varying concentrations of ERGi-USU. The compound quenched the fluorescence of HsRIOK2 in a concentration-dependent manner (0.67 to 670 nmol/L). Data fitting with a two-site binding model yielded a high-affinity site Kd of 64 ± 30 nmol/L. [1]
The same assay was performed using purified Riok2 from Chaetomium thermophilum (CtRiok2). ERGi-USU showed much lower affinity for CtRiok2, with quenching observed only at 6.7 µmol/L and above. Data fitting with a one-site model yielded a Kd of 1.3 ± 0.6 µmol/L, indicating at least 10-fold higher affinity for human RIOK2. [1]

For the primary screen identifying ERGi-USU, an In-Cell Western assay was used. VCaP cells were plated in 96-well plates and treated with library compounds at 1 µmol/L for 48 hours. Cells were then washed, fixed, permeabilized, and immunolabeled with an anti-ERG primary antibody. After washing, cells were stained with a non-vital cell stain, a DNA stain, and a secondary antibody conjugated to a fluorescent dye. Plates were scanned, and fluorescence intensities for ERG protein and cell density were measured. Ratios were normalized, and compounds causing a decrease greater than 2.0 standard deviations from the mean in duplicate experiments were selected. [1]
For cell growth inhibition assays, cells were incubated with indicated concentrations of ERGi-USU for 8 days. Cells were then trypsinized and counted manually using a hemocytometer and trypan blue staining. IC50 values were calculated from dose-response curves. [1]
For Western blot analysis, cells were treated with ERGi-USU for specified times (e.g., 48 hours), harvested, and lysed. Lysates were separated by SDS-PAGE, transferred to membranes, and probed with primary antibodies against target proteins (e.g., ERG, RIOK2, PARP, caspases) and loading controls (e.g., GAPDH, α-tubulin), followed by appropriate secondary antibodies. [1]
For combination studies with enzalutamide, VCaP cells were treated with varying doses of ERGi-USU alone or in combination with enzalutamide, and cell growth was assessed. [1]

To evaluate the antitumor activity of ERGi-USU in vivo, VCaP cells were subcutaneously injected into male nude mice to establish xenograft tumors. [1]
When tumors became palpable, mice were randomized into treatment groups. ERGi-USU was administered intraperitoneally at doses of 100 mg/kg or 150 mg/kg, three times per week. The control group received vehicle only. [1]
Tumor dimensions were measured regularly, and tumor volumes were calculated. Body weight was monitored as an indicator of toxicity. The experiment was terminated, and tumors were excised and weighed at the end of the study period. [1]

In nude mice carrying VCaP xenografts, no significant systemic toxicity (e.g., weight loss, somnolence, diarrhea, loss of appetite, dyspnea) was observed after intraperitoneal injection of ERGi-USU (at doses of 100 mg/kg and 150 mg/kg, three times a week). No damage was found on gross examination of major organs. [1]
Local inflammation was occasionally observed at the injection site in the 150 mg/kg dose group. [1]
This article cites previous data from NCI-DTP indicating that NSC139021 (ERGi-USU) was non-toxic to mice in the dose range of 12.5 to 400 mg/kg. [1]

[1]. Identification of a small molecule that selectively inhibits ERG-positive cancer cell growth. Cancer Res. 2018 Apr 30. pii: canres.2949.2017.

ERGi-USU, also known as NSC139021 and 1-[2-thiazolylazo]-2-naphthol, was selected from 2407 small molecule compounds that are inhibitors of ERG oncoprotein expression in VCaP prostate cancer cells. [1] It is highly selective in inhibiting the growth of ERG-positive cancer cells (prostate cancer, colon cancer, leukemia) while having minimal effect on normal endothelial cells (HUVEC) and ERG-negative cells, indicating that it has a high therapeutic index. [1] Its mechanism of action involves directly binding to and inhibiting the atypical kinase RIOK2, leading to disordered ribosome biosynthesis, ribosome stress induction, and subsequent downregulation of ERG protein, ultimately triggering apoptosis and cell cycle arrest in ERG-positive cancer cells. [1] When used in combination with the androgen receptor inhibitor enzalutamide, this compound inhibits the growth of ERG-positive VCaP cells, showing a synergistic effect. [1]
Its core structure consists of a thiazole ring, a diazo linkage, and a naphthol ring. Modification of the thiazole ring or the diazo linkage eliminates activity, while replacing naphthol with a 5-alkyl-substituted phenolic compound retains activity. [1]

C13H9N3OS

255.2951

255.047

1147-56-4

93572

Pink to red solid powder

1.4g/cm3

523.251ºC at 760 mmHg

138-139 °C

270.253ºC

0mmHg at 25°C

1.728

4.417

1

5

2

18

313

0

S1C([H])=C([H])N=C1/N=N/C1=C(C([H])=C([H])C2=C([H])C([H])=C([H])C([H])=C21)O[H]

IOMXCGDXEUDZAK-UHFFFAOYSA-N

InChI=1S/C13H9N3OS/c17-11-6-5-9-3-1-2-4-10(9)12(11)15-16-13-14-7-8-18-13/h1-8,17H

1-(1,3-thiazol-2-yldiazenyl)naphthalen-2-ol

NSC-139021; ERGi-USU; NSC 139021; ERGi USU; NSC139021; ERGiUSU;

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 mg/mL (~242.85 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.79 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 (9.79 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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 (Please use freshly prepared in vivo formulations for optimal results.)