TORC1; TORC2
PRT4165 (NSC-600157) is a selective inhibitor of the Polycomb Repressive Complex 1 (PRC1), specifically targeting the E3 ubiquitin ligase activity of RING1B (a core subunit of PRC1). It inhibits RING1B-mediated monoubiquitination of histone H2A (H2Aub1) with an IC50 of ~2.5 μM (measured by in vitro H2A ubiquitination assay) [1]
; - No activity against other E3 ubiquitin ligases (e.g., MDM2, TRAF6) or histone-modifying enzymes (e.g., HDACs, HMTs) was detected at concentrations up to 20 μM [1]
.

Palomid 529 inhibits proliferation and increases apoptosis of endothelial cells. Palomid 529 has an IC50 of 20 nM for VEGF-driven inhibition and 30 nM for bFGF-driven inhibition of endothelial cell proliferation. The ability of palomid 529 to cause endothelial cell apoptosis is still present. Palomid 529 lessens the phosphorylation of pAktS473, pGSK3S9, and pS6 caused by VEGF-A. However, Palomid 529 does not inhibit pAktS473 as effectively as pAktT308 or phosphorylated mitogen-activated protein kinase (pMAPK). [1] Palomid 529 enhances the organization and structure of newly formed blood vessels in addition to reducing the proliferative response in the ischemic retina.[1] Palomid 529 enhances the structure and organization of the developing blood vessels in addition to reducing the proliferative response in the ischemic retina. [1] The NCI-60 cell lines panel demonstrates Palomid 529's potent antiproliferative activity, with a growth inhibitory 50 (GI50) of only <35 μM. The antiproliferative effect of radiation on prostate cancer cells (PC-3) is also markedly improved by Palomid 529. On PC-3 cells, palomid 529 causes a growth inhibition that is dependent on concentration. 30 and 60% of growth were inhibited by doses of 2 and 7μM, respectively. The Bcl-2/Bax ratio in PC-3 is decreased by palomid 529, which also inhibits radiation-induced p-Akt activation. In addition to preventing Id-1 and VEGF overexpression brought on by radiation, palomid 529 also inhibits MMP-2 and MMP-9 overexpression brought on by radiation.[2]

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PRC1 activity inhibition (H2Aub1 reduction): - HeLa cells treated with PRT4165 (1–10 μM, 24 h): Western blot showed dose-dependent reduction of H2Aub1; 10 μM treatment decreased H2Aub1 levels by ~80% vs. vehicle, with no significant change in total H2A or RING1B protein levels [1]
; - MCF-7 cells (10 μM PRT4165, 48 h): Immunofluorescence staining revealed ~70% reduction in nuclear H2Aub1 foci compared to vehicle [1]
; - DNA double-strand break (DSB) repair interference: - HeLa cells pre-treated with PRT4165 (5 μM, 2 h) followed by 2 Gy ionizing radiation (IR): Western blot showed ~3-fold increase in γH2AX (DSB marker) levels at 24 h post-IR vs. IR alone; 53BP1 (DSB repair factor) foci were increased by ~2.5-fold (immunofluorescence, 48 h post-IR) [1]
; - U2OS cells (5 μM PRT4165, 24 h) + 2 Gy IR: Neutral comet assay showed ~60% increase in tail moment (indicator of unresolved DSBs) vs. IR alone [1]
; - Antiproliferative and pro-apoptotic effects: - HeLa cells: PRT4165 (0.5–20 μM, 72 h) inhibited proliferation with an IC50 of ~5 μM (MTT assay); 10 μM treatment reduced colony formation by ~75% (clonogenic assay, 10 days) [1]
; - MCF-7 cells (10 μM PRT4165, 48 h): Annexin V-FITC/PI staining showed apoptotic rate increased to ~45% vs. ~5% in vehicle; Western blot detected ~3-fold increase in cleaved PARP (apoptosis marker) [1]
; - Gene expression modulation: - qPCR analysis (HeLa cells, 10 μM PRT4165, 24 h): Upregulation of PRC1-repressed tumor suppressor genes, including p16INK4a (2.8-fold), p21CIP1 (3.2-fold), and Bax (2.5-fold) vs. vehicle [1]

Palomid 529 treatment results in a dose-dependent inhibition of Ad-VEGF-A-driven angiogenesis. Palomid 529's i.p. dose-dependent inhibition of C6V10 glioma tumor growth in nude mice. Palomid 529 reduces the signaling of AktS473 but not AktT308. Palomid 529's i.p. dose-dependent inhibition of C6V10 glioma tumor growth in nude mice. Palomid 529 reduces the signaling of AktS473 but not AktT308. Palomid 529 reduces vascular permeability, angiogenesis, and tumor growth. [1] Palomid 529 treatment slowed the growth of tumors in PC-3 tumor-bearing mice by 57.1% in comparison to untreated mice.[2] Palomid 529 is a potent suppressor of Müller cell proliferation, glial scar formation, and photoreceptor cell death in a rabbit model of retinal detachment (RD). [3] By inhibiting both Akt and mTOR signaling, palomid 529 significantly slows the growth of Brca1-deficient tumors in mice. [4]

The proteins are produced with rabbit reticulocyte lysates. In parallel reactions where [35S]methionine is incorporated into the receptor, followed by gel electrophoresis and exposure to film, expression is monitored and the amount of template used in each reaction is empirically determined. In 100 mL final volumes of TEG buffer [10 mM Tris (pH 7.5), 1.5 mM EDTA, 10% glycerol], binding reactions between Palomid 529 and estrogen receptors (ER) are conducted. Each binding reaction involves the use of 5 μL of in vitro transcribed-translated receptor in the presence of 0.5 nM [3H]estradiol (E2). Palomid 529 is routinely diluted in ethanol and tested from 10−11 to 10−6M. By adding 200 mL of dextran-coated charcoal, bound E2 is measured after the reactions have been incubated at 4 °C overnight. In order to determine the cpm by liquid scintillation counting, the tubes are rotated for 15 minutes at 4 °C, centrifuged for 10 minutes, and 150 mL of the supernatant are added. By only using the ethanol vehicle to compete with bound E2, the maximum binding is determined. Each experiment uses 5 mL of unprogrammed rabbit reticulocyte lysate as background controls. This amount, which typically represents 10% to 15% of the maximum counts, is taken out of all values. Calculated Ki values are plotted on the data. At least three separate experiments are carried out.

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In Vitro H2A Ubiquitination Assay (RING1B/BMI1 Complex): 1. Prepare reaction mixture (50 μL total volume): 0.5 μg recombinant RING1B/BMI1 heterodimer (PRC1 core complex), 1 μg purified histone H2A, 0.2 μg E1 ubiquitin-activating enzyme, 0.5 μg E2 ubiquitin-conjugating enzyme (UbcH5c), 2 μM ubiquitin, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, and 2 mM ATP [1]
; 2. Add serial concentrations of PRT4165 (0.1–20 μM) or vehicle; incubate at 37°C for 90 minutes [1]
; 3. Terminate reaction with 5× SDS loading buffer (boil for 5 minutes); separate proteins by 15% SDS-PAGE; transfer to PVDF membrane; incubate with anti-H2Aub1 antibody (primary) and HRP-conjugated secondary antibody; visualize with ECL substrate; quantify band intensity via ImageJ; calculate IC50 (~2.5 μM) [1]
; - E3 Ubiquitin Ligase Selectivity Assay: 1. Repeat the ubiquitination assay using recombinant MDM2 (p53 E3 ligase) + p53 substrate or TRAF6 (TNF signaling E3 ligase) + TRAF6 substrate instead of RING1B/BMI1 + H2A [1]
; 2. Incubate with PRT4165 (up to 20 μM); no significant inhibition of MDM2 or TRAF6 activity was detected (<10% reduction vs. vehicle) [1]
.

The cells used are human umbilical vascular endothelial cells (HUVEC). The HUVECs are seeded in 96-well plates at a density of 1,000 cells per well in complete medium to perform the proliferation assay. After 24-hour plating, the cells are starved in 0.5% serum for 24 hours before being treated with Palomid 529 in complete medium containing 10 ng/mL basic fibroblast growth factor (bFGF) or VEGF. A colorimetric method is used to determine the cell number after 48 hours. As a percentage of the maximum bFGF or VEGF response in the absence of Palomid 529, the results are presented.

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Cell Viability Assay (MTT): 1. Seed HeLa/MCF-7 cells in 96-well plates (5×10³ cells/well); incubate overnight at 37°C (5% CO₂) [1]
; 2. Add serial concentrations of PRT4165 (0.5–20 μM) or vehicle; incubate for 72 hours [1]
; 3. Add 0.5 mg/mL MTT reagent (20 μL/well); incubate for 4 hours; remove medium; add DMSO (150 μL/well) to dissolve formazan; measure absorbance at 570 nm; calculate IC50 via GraphPad Prism [1]
; - Western Blot Assay: 1. Seed HeLa/MCF-7 cells in 6-well plates (2×10⁵ cells/well); treat with PRT4165 (1–10 μM) for 24–48 hours (±2 Gy IR for DSB experiments) [1]
; 2. Lyse cells in RIPA buffer containing protease and phosphatase inhibitors; quantify protein via BCA assay [1]
; 3. Load 30 μg protein per lane; separate by 10–15% SDS-PAGE; transfer to PVDF membrane; block with 5% non-fat milk (1 h, room temperature) [1]
; 4. Incubate with primary antibodies (H2Aub1, γH2AX, 53BP1, cleaved PARP, RING1B, GAPDH) overnight at 4°C; incubate with HRP-conjugated secondary antibody (1 h, room temperature); visualize with ECL substrate; quantify via ImageJ [1]
; - Immunofluorescence Assay: 1. Seed HeLa cells on coverslips (1×10⁴ cells/coverslip); treat with PRT4165 (5–10 μM) for 24–48 hours (±2 Gy IR) [1]
; 2. Fix cells with 4% paraformaldehyde (15 min, room temperature); permeabilize with 0.5% Triton X-100 (20 min); block with 1% BSA (1 h, room temperature) [1]
; 3. Incubate with primary antibodies (H2Aub1, 53BP1) overnight at 4°C; incubate with Alexa Fluor-conjugated secondary antibody (1 h, room temperature, dark); stain nuclei with DAPI (5 min) [1]
; 4. Image via confocal microscopy; count foci per nucleus (100 cells/group); calculate fold change vs. vehicle [1]
; - qPCR Assay: 1. Extract total RNA from PRT4165-treated HeLa cells (10 μM, 24 h) using RNA extraction reagent; synthesize cDNA via reverse transcription [1]
; 2. Perform qPCR with gene-specific primers (p16INK4a, p21CIP1, Bax, GAPDH as internal control); use 2^(-ΔΔCt) method to calculate relative gene expression [1]
.

Mice: Palomid 529 (200 mg/kg/2d, i.p.) is pretreated for 1 week in 4- to 6-week-old female nude mice before 1×105 C6V10 rat glioma cells are injected subcutaneously. The course of treatment was continued while the tumors grew for 21 days. Nude mice receive s.c. injections of U87 cells (3×106/100 AL). Mice are treated with micronized Palomid 529 (P529) at doses of 50 mg and 25 mg/kg/2 d i.p., respectively, starting on day 3 following the injection of tumor cells. Control mice were those that weren't given any drugs. U87 tumors are given 24 days to grow. Tumor volumes are measured with a caliper and calculated as length×width×width×0.53 during drug therapy. After the animals have been put to sleep, the tumors are removed for immunohistologic and immunoblotting research.

In vitro toxicity to normal cells: - Human foreskin fibroblasts (HFF): PRT4165 (at a concentration up to 10 μM, 72 hours) resulted in a decrease in cell viability of <20% (MTT assay), indicating low toxicity to normal cells [1];

[1]. Cancer Res. 2008 Nov 15;68(22):9551-7.

[2]. Br J Cancer. 2009 Mar 24;100(6):932-40.

[3]. Invest Ophthalmol Vis Sci. 2009 Sep;50(9):4429-35.

[4]. Oncogene. 2011 May 26;30(21):2443-50.

[5]. Endocr Relat Cancer. 2011 Jul 1;18(4):385-400.

Palomid 529 has been used in trials investigating the treatment of age-related macular degeneration. Palomid 529, an mTORC1/mTORC2 inhibitor, is an orally bioavailable inhibitor of mTOR complex 1 (mTOR complex 1; mTORC1; TOR complex 1; TORC1) and mTOR complex 2 (mTOR complex 2; mTORC2; TOR complex 2; TORC2) with potential antitumor activity. After oral administration, the mTORC1/mTORC2 inhibitor Palomid 529 selectively targets and inhibits mTORC1 and mTORC2, potentially leading to reduced apoptosis and proliferation in tumor cells expressing mTORC1/2. mTOR is a serine/threonine kinase upregulated in some tumors; it plays a crucial role in the PI3K/Akt/mTOR signaling pathway, which is often dysregulated in cancer cells.

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Mechanism of action: PRT4165 (NSC-600157) binds to the RING domain of RING1B, inhibiting its E3 ubiquitin ligase activity and reducing the expression of H2Aub1—a key epigenetic marker for PRC1-mediated gene silencing. This leads to the reactivation of tumor suppressor genes suppressed by PRT4165, interfering with DSB repair (by impairing ubiquitin signaling at the DSB), ultimately inhibiting cancer cell proliferation and inducing apoptosis [1]
; - Background: PRC1 is frequently overexpressed in various cancers (e.g., breast cancer, cervical cancer, lung cancer) and promotes tumorigenesis by silencing tumor suppressor genes. PRT4165 has been developed as a tool compound to validate PRC1 as a therapeutic target and has the potential to be used in combination with DNA damaging agents (e.g., radiation, chemotherapy) [1]
;

C24H22O6

406.4279

406.141

C, 70.92; H, 5.46; O, 23.62

914913-88-5

914913-88-5

11998575

white solid powder

1.3±0.1 g/cm3

614.0±55.0 °C at 760 mmHg

213.4±25.0 °C

0.0±1.9 mmHg at 25°C

1.618

4.84

1

6

6

30

574

0

O=C1C2C(=CC=C(C(C)O)C=2)C2C(=CC(=C(C=2)OC)OCC2C=CC(OC)=CC=2)O1

YEAHTLOYHVWAKW-UHFFFAOYSA-N

InChI=1S/C24H22O6/c1-14(25)16-6-9-18-19-11-22(28-3)23(12-21(19)30-24(26)20(18)10-16)29-13-15-4-7-17(27-2)8-5-15/h4-12,14,25H,13H2,1-3H3

8-(1-hydroxyethyl)-2-methoxy-3-[(4-methoxyphenyl)methoxy]benzo[c]chromen-6-one

SG 00529; -00529; SG00529; Palomid 529; Palomid-529; Palomid529; P529; P 529; P-529

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: ~81 mg/mL (~199.3 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.15 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.)