RA190 covalently binds to cysteine 88 of the ubiquitin receptor RPN13 in 19S regulatory granules, inhibits proteasome action, and induces the fast accumulation of polyubiquitinated proteins. Multiple myeloma (MM) lines, especially those resistant to bortezomib, are sensitive to RA190 through endoplasmic reticulum stress-associated apoptosis. RA190 stabilizes the target of the human papillomavirus (HPV) E6 oncoprotein and preferentially kills HPV-transformed cells [1].

RA190 covalently binds to cysteine 88 of the ubiquitin receptor RPN13 in 19S regulatory granules, inhibits proteasome action, and induces the fast accumulation of polyubiquitinated proteins. Multiple myeloma (MM) lines, especially those resistant to bortezomib, are sensitive to RA190 through endoplasmic reticulum stress-associated apoptosis. RA190 stabilizes the target of the human papillomavirus (HPV) E6 oncoprotein and preferentially kills HPV-transformed cells [1].

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RA190 exhibits potent anti-proliferative activity against multiple myeloma (MM) cell lines (IC50 ~0.1 μM) and human papillomavirus (HPV)-transformed cervical cancer cell lines (HeLa, CaSki, SiHa; IC50 ~0.3 μM), while being less effective against HPV-negative cervical cancer lines (IC50 >5 μM for HT3 and C33A). [1]
RA190 is equally efficacious against bortezomib-resistant MM cell lines (ANBL6-V10R, RPMI-8226-V10R) and their parental lines. [1]
Treatment with RA190 leads to rapid, dose-dependent accumulation of high molecular weight K48-linked polyubiquitinated proteins in HeLa and CaSki cells, occurring more rapidly and with a different profile compared to bortezomib. [1]
RA190 does not inhibit the chymotrypsin-like, trypsin-like, or PGPH (peptidylglutamyl-peptide hydrolyzing) activities of the 20S core particle (CP) of the proteasome. [1]
RA190 minimally impacts the deubiquitinase activity of purified recombinant UCH37 or the 19S regulatory particle (RP) in an Ub-AMC cleavage assay. [1]
Using a tetraubiquitin-fused luciferase (4UbFL) reporter assay in HeLa cells, RA190 stabilizes the reporter, confirming proteasome inhibition in live cells. [1]
RA190 treatment upregulates endoplasmic reticulum (ER) stress markers: increased ATF-4 protein levels, and elevated mRNA levels of CHOP-10 and spliced XBP1 (XBP1s) in MM.1S and HeLa cells. Bax protein levels are also elevated. [1]
In HPV+ cervical cancer cells (HeLa, CaSki, SiHa), RA190 treatment stabilizes the HPV E6 oncoprotein targets p53, p21, Puma, Bax, Bak, and hDLG-1 in a time-dependent manner. [1]
RA190 induces apoptosis in MM and HeLa cells, as evidenced by Annexin V staining, activation of caspase-3/7, and cleavage of PARP. [1]
RA190 treatment induces cell surface display of HSP90 on HeLa cells, a phenomenon associated with immunogenic cell death. [1]
Chemical modifications that eliminate the Michael acceptor properties of RA190 (e.g., RA190ME, RA190R) significantly reduce or abolish its cell-killing activity and ability to induce polyubiquitin accumulation. [1]

RA190 inhibits the activity of proteasomes in skin and muscle and is distributed to plasma and major organs, excluding the brain. Multiple myeloma and ovarian cancer xenografts grow much slower when RA190 is administered, and oral RA190 treatment slows the growth of tumors in HPV16+ syngeneic mice without altering the spontaneous responses of HPV-specific CD8+ T cells [1].

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Intraperitoneal (i.p.) or oral (p.o.) administration of RA190 inhibits proteasome function in mouse skin and muscle, as demonstrated by stabilization of a 4UbFL reporter delivered by gene gun or electroporation. [1]
Daily i.p. treatment with 20 mg/kg RA190 for 7 days potently inhibited the growth of NCI-H929 multiple myeloma xenografts in NOG mice. [1]
Daily i.p. treatment with 10 mg/kg RA190 for 14 days significantly inhibited the growth of ES2 ovarian cancer xenografts in nude mice. [1]
Oral administration of RA190 (40 mg/kg every third day) significantly inhibited the growth of syngeneic TC-1 (HPV16+) tumors in C57BL/6 mice without affecting the spontaneous E7-specific CD8+ T cell response or animal weight gain. [1]
Tumor lysates from RA190-treated TC-1 tumor-bearing mice showed dramatically elevated levels of polyubiquitinated proteins. [1]
Topical administration of 4% RA190 in Cremophor-EL stabilized the 4UbFL reporter in mouse skin. [1]

The 20S proteasome catalytic activity assay measures the chymotrypsin-like, trypsin-like, and PGPH activities. Cell lysates or purified 20S proteasomes are incubated with specific fluorogenic peptide substrates (e.g., Suc-LLVY-AMC for chymotrypsin-like activity) in reaction buffer. The release of free AMC is monitored by fluorescence spectrophotometry. RA190 was tested in this assay and showed no inhibition of these three catalytic activities. [1]
The deubiquitinase activity assay assesses the activity of the 19S regulatory particle (RP) or recombinant UCH37. Purified 19S RP or recombinant UCH37 (with or without RPN13) is incubated with ubiquitin-AMC (Ub-AMC) substrate in an appropriate assay buffer. The release of free AMC, indicative of deubiquitinase activity, is measured by fluorescence. RA190 showed minimal impact on this activity. [1]

Cell viability/anti-proliferative activity was determined using an XTT assay. Cancer cell lines were seeded in 96-well plates and treated with titrated concentrations of compounds for 48 hours. The XTT reagent was then added, and after incubation, absorbance was measured to determine the percentage of viable cells relative to untreated controls. IC50 values were calculated from dose-response curves. [1]
For polyubiquitinated protein accumulation, cells were treated with compounds for specified times (e.g., 4 or 12 hours). Cells were then lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with an anti-K48-linked ubiquitin antibody. β-tubulin or actin immunoblotting was used as a loading control. [1]
The tetraubiquitin-fused luciferase (4UbFL) reporter assay was performed to assess proteasome function in live cells. HeLa cells were transiently transfected with either 4UbFL or unfused firefly luciferase (FL) expression plasmids. After 48 hours, cells were treated with compounds for 4 hours. Luciferase activity was measured, and the ratio of bioluminescence in 4UbFL-transfected cells to FL-transfected cells was calculated to indicate proteasome inhibition. [1]
Apoptosis was assessed by flow cytometry using an Annexin V-PE staining kit according to the manufacturer's protocol. Cells were treated with compounds, harvested, stained with Annexin V-PE and 7-AAD (or similar viability dye), and analyzed by flow cytometry. [1]
Caspase-3/7 activity was measured by flow cytometry using an antibody specific for active caspase-3. Treated cells were fixed, permeabilized, stained with the antibody, and analyzed by flow cytometry. [1]
Western blot analysis for apoptotic markers (e.g., PARP cleavage) and ER stress/UPR markers (e.g., ATF-4, Bax) was performed. Treated cells were lysed, proteins were separated by SDS-PAGE, transferred to membranes, and probed with specific antibodies. [1]
Cell surface HSP90 display was analyzed by flow cytometry. Treated cells were harvested, stained with an anti-HSP90 antibody without permeabilization, followed by a PE-conjugated secondary antibody, and analyzed by flow cytometry. [1]
Quantitative PCR (q-PCR) was used to measure mRNA levels of CHOP-10 and XBP1s. Total RNA was extracted from treated cells, reverse transcribed, and subjected to q-PCR using specific primers and probes. mRNA levels were normalized to GAPDH and expressed as fold change over control. [1]

For pharmacokinetic studies, mice were administered a single dose of RA190 (10 mg/kg i.p. or 20 mg/kg p.o.) formulated in 20% (w/v) β-hydroxylsopropyl-cyclodextrin in water. Blood samples were collected at various time points post-dose via retro-orbital bleeding or cardiac puncture. Plasma was separated, and RA190 concentrations were determined by LC-MS/MS. For tissue distribution, organs were harvested 48 hours post-i.p. dosing and analyzed for RA190 content. [1]
For proteasome inhibition assessment in vivo, mice received the 4UbFL reporter plasmid delivered either intramuscularly (i.m.) by electroporation (eight pulses at 106 V for 20 ms) or intradermally via helium-driven gene gun. One day later, mice were treated with RA190 (40 mg/kg i.p. or p.o.), bortezomib (1.5 mg/kg i.p.), vehicle, or topical 4% RA190 in Cremophor-EL. Bioluminescence was measured by IVIS imaging after luciferin injection at specified times post-treatment. [1]
For multiple myeloma xenograft efficacy, NOG mice were inoculated intravenously with NCI-H929-GFP-Luc cells. After tumor establishment (4 weeks), mice were treated with RA190 (20 mg/kg/day i.p.) or vehicle for 7 days. Tumor growth was monitored by bioluminescence imaging before and after treatment. [1]
For ovarian cancer xenograft efficacy, nude mice were inoculated intraperitoneally with ES2-luciferase cells. Three days later, mice were treated with RA190 (10 mg/kg/day i.p.) or vehicle for 14 days. Tumor burden was assessed by weekly bioluminescence imaging. [1]
For syngeneic HPV+ tumor efficacy, C57BL/6 mice were challenged subcutaneously with TC-1 cells. When tumors were palpable, mice were treated orally with RA190 (40 mg/kg every third day) or vehicle for 14 days. Tumor volume was measured regularly. At the endpoint, tumors were weighed, and splenocytes were harvested to assess E7-specific CD8+ T cell responses by intracellular cytokine staining (IFN-γ) after peptide stimulation. [1]
For toxicity assessment, tumor-free mice were treated orally with 40 mg/kg RA190 or vehicle every third day for three doses and euthanized on day 12. Blood was collected for hematology and clinical chemistry analysis. Major organs (lungs, kidney, spleen, liver) were harvested for histopathological examination. [1]

Following a single intraperitoneal injection of 10 mg/kg RA190 in mice, peak plasma concentration (Cmax) was reached at 2 hours (Tmax). The distribution half-life (T1/2, α) was 4.2 hours, and the terminal half-life (T1/2, β) was 25.5 hours. [1] Following a single oral administration of 20 mg/kg RA190, plasma concentrations rapidly increased within 1 hour, with a terminal half-life (T1/2, β) of 2.6 hours. [1] Based on AUC values, the oral bioavailability of RA190 is approximately 7.2% relative to intraperitoneal administration. [1]
48 hours after a single intraperitoneal injection of 10 mg/kg, RA190 was detected in the kidneys (0.61 µg/g), liver (0.57 µg/g), lungs (0.66 µg/g), and spleen (0.59 µg/g), but not in brain tissue. [1]

Mice were orally administered RA190 at 40 mg/kg every three days for three consecutive times. Compared with the vector control group, except for a slight decrease in triglyceride levels, there were no significant differences in hematological parameters (complete blood cell count) or clinical chemical indicators. [1]
Histopathological examination of the lungs, kidneys, spleens and livers of mice in the RA190 treatment group showed no significant abnormalities compared with the control group. [1]
In TC-1 tumor-bearing mice, RA190 treatment had no significant effect on weight gain compared with the vector control group. [1]

[1]. A bis-benzylidine piperidone targeting proteasome ubiquitin receptor RPN13/ADRM1 as a therapy for cancer. Cancer Cell. 2013 Dec 9;24(6):791-805.

RA190 is a bisbenzylpiperidone derivative designed for the Michael receptor. Its α,β-unsaturated ketone (enone) moiety is the minimum pharmacophore required to exert its activity. [1]
Its mechanism of action involves covalent binding to the C88 site of the ubiquitin receptor RPN13 in the proteasome 19S regulatory granule. This inhibits the function upstream of the proteasome catalytic core, leading to rapid accumulation of polyubiquitinated proteins, endoplasmic reticulum stress, unfolded protein response (UPR), and apoptosis. [1]
RA190 has shown synergistic effects with bortezomib in killing HeLa cells in vitro, suggesting that the two have different targets and potential for combination therapy. [1]
This drug has shown potential for treating multiple myeloma (including bortezomib-resistant types), HPV-related cancers (e.g., cervical cancer), and ovarian cancer. [1]
Unlike bortezomib and carfilzomib, which target the β subunit of the 20S core particle catalytic structure, RA190 targets the regulatory subunit RPN13, providing a different mechanism that may overcome certain resistance and reduce off-target side effects such as neuropathy and thrombocytopenia. [1]
RPN13 is frequently amplified in ovarian and colon cancers, suggesting a cancer-specific vulnerability that RA190 may exploit. [1]

C28H23CL5N2O2

596.7594

596.017

1617495-03-0

1617572-02-7;1617495-03-0 (HCl);

126843229

Light yellow to yellow solid powder

2

3

5

37

809

1

ClC1=C(C([H])=C([H])C(=C1[H])/C(/[H])=C1\C(/C(=C(/[H])\C2C([H])=C([H])C(=C(C=2[H])Cl)Cl)/C([H])([H])N(C([C@]([H])(C([H])([H])C2C([H])=C([H])C([H])=C([H])C=2[H])N([H])[H])=O)C\1([H])[H])=O)Cl.Cl[H]

UMWXLEVUBFNYIK-VCCJZKHWSA-N

InChI=1S/C28H22Cl4N2O2.ClH/c29-22-8-6-18(12-24(22)31)10-20-15-34(28(36)26(33)14-17-4-2-1-3-5-17)16-21(27(20)35)11-19-7-9-23(30)25(32)13-19;/h1-13,26H,14-16,33H2;1H/b20-10-,21-11-;/t26-;/m0./s1

(3Z,5Z)-1-[(2S)-2-amino-3-phenylpropanoyl]-3,5-bis[(3,4-dichlorophenyl)methylidene]piperidin-4-one;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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 (~167.57 mM)

Solubility in Formulation 1: ≥ 2.38 mg/mL (3.99 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 23.8 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.)