Biological Activity

Bioactivity / Mechanism

ONX-0914 (PR-957) targets the immunoproteasome. Its selectivity depends on exposure and assay context: purified human immunoproteasomes showed LMP2 inhibition, and mouse lymphocytes exposed to 300 nM for 2 h showed modification of both LMP7 and LMP2. These data limit use of ONX-0914 as an exclusively LMP7-selective probe. [1]

Literature-reported observations and conditions; not Adooq-tested protocols or general treatment recommendations.

Target & Activity

Target	Activity	Value
LMP7 (Cell-free assay)		~10 nM

Cellular Effect

Cell Line	Activity	Value	Description	Reference
Caco-2	EC50	1.3 μM	Cytotoxicity against human Caco2 cells assessed as reduction in cell viability after 48 hrs by Hoechst 33342 staining based HCS assay	[PMID: 29339252]
Fibroblast	EC50	0.16 μM	Cytotoxicity against human fibroblasts assessed as reduction in cell viability after 48 hrs by Hoechst 33342 staining based HCS assay	[PMID: 29339252]
HCT-116	EC50	0.11 μM	Cytotoxicity against human HCT116 cells assessed as reduction in cell viability after 48 hrs by Hoechst 33342 staining based HCS assay	[PMID: 29339252]
Huh-7	EC50	0.39 μM	Cytotoxicity against human HuH7 cells assessed as reduction in cell viability after 48 hrs by Hoechst 33342 staining based HCS assay	[PMID: 29339252]
MCF7	EC50	0.26 μM	Cytotoxicity against human MCF7 cells assessed as reduction in cell viability after 48 hrs by Hoechst 33342 staining based HCS assay	[PMID: 29339252]
MDA-MB-231	EC50	0.27 μM	Cytotoxicity against human MDA-MB-231 cells assessed as reduction in cell viability after 48 hrs by Hoechst 33342 staining based HCS assay	[PMID: 29339252]
MM1.S	IC50	0.24 μM	Antiproliferative activity against human MM1.S cells assessed as inhibition of cell proliferation after 72 hrs by MTS assay	[PMID: 34087498]
NCI-H727	EC50	0.3 μM	Cytotoxicity against human NCI-H727 cells assessed as reduction in cell viability after 48 hrs by Hoechst 33342 staining based HCS assay	[PMID: 29339252]
PBMC	IC50	0.12 μM	Anti-inflammatory activity in human PBMC assessed as inhibition of LPS-induced IL-12/23 p40 production pretreated for 1 hr followed by LPS stimulation measured after 20 hrs	[PMID: 28435528]
PBMC	IC50	0.22 μM	Anti-inflammatory activity in human PBMC assessed as inhibition of LPS-induced GMCSF production pretreated for 1 hr followed by LPS stimulation measured after 20 hrs	[PMID: 28435528]
PBMC	IC50	0.24 μM	Anti-inflammatory activity in human PBMC assessed as inhibition of LPS-induced IL-6 production pretreated for 1 hr followed by LPS stimulation measured after 20 hrs	[PMID: 28435528]
PBMC	IC50	0.56 μM	Anti-inflammatory activity in human PBMC assessed as inhibition of LPS-induced TNF-alpha production pretreated for 1 hr followed by LPS stimulation measured after 20 hrs	[PMID: 28435528]
PBMC	IC50	1.11 μM	Anti-inflammatory activity in human PBMC assessed as inhibition of LPS-induced IL-8 production pretreated for 1 hr followed by LPS stimulation measured after 20 hrs	[PMID: 28435528]
PBMC	IC50	1.47 μM	Anti-inflammatory activity in human PBMC assessed as inhibition of LPS-induced Il-1b production pretreated for 1 hr followed by LPS stimulation measured after 20 hrs	[PMID: 28435528]
PBMC	IC50	3.4 μM	Cytotoxicity against human PBMC assessed as decrease in cell viability by CellTiter-Glo assay	[PMID: 28435528]
PC-3	EC50	0.33 μM	Cytotoxicity against human PC3 cells assessed as reduction in cell viability after 48 hrs by Hoechst 33342 staining based HCS assay	[PMID: 29339252]
Raji	IC50	> 10 μM	Inhibition of 20s proteasome subunit beta-1c in human Raji cells using BODIPY- epoxomicin by fluorescent densitometry	[PMID: 25006746]
Raji	IC50	0.46 μM	Inhibition of proteasome subunit beta-1i in human Raji cells using BODIPY- epoxomicin by fluorescent densitometry	[PMID: 25006746]
Raji	IC50	0.59 μM	Inhibition of proteasome subunit beta-2i in human Raji cells using BODIPY- epoxomicin by fluorescent densitometry	[PMID: 25006746]

19 records · Scroll to view more

In Vitro Studies

In 3T3-L1 adipocyte differentiation, ONX-0914 at 1–2 μM during the first two days reduced final lipid accumulation. Exposure during the later maintenance phase showed a reduction tendency that was not statistically significant. A day-1 viability control did not establish absence of longer-term cellular stress. [2]

Human immunoproteasome LMP2 inhibition

Biochemical assay · Purified human 20S immunoproteasome [1]

ONX 0914 inhibited LMP2 substrate cleavage; PRN1126 did not alter cleavage up to 10 μM. This endpoint limits an exclusively LMP7-selective interpretation.

Measured endpoints — not treatment concentrations
Endpoint	Reported result	Reference
Human LMP2-mediated Ac-PAL-AMC cleavage	IC50 1.1 ± 0.5 × 10^−7 M	[1]

Literature-Reported Treatment Conditions

Method: Concentration-response fluorogenic Ac-PAL-AMC hydrolysis assay; substrate 12.5 μM.
Vehicle: DMSO; shared Methods specify final 0.3% for in vitro experiments.
Reported limits: Assay-specific preincubation/duration is not specified in the retrieved main-text assay description; no timing borrowed from cell assays. Concentrations are plotted in Figure 3A; IC50 is an endpoint, not a treatment dose.

Methods and source locations

Compound reported in the paper: ONX-0914; Compound label in the paper: ONX 0914 (PR-957)

Applies to named ONX 0914/PR-957 in the stated model; counterion and lot equivalence are not independently established. No salt conversion or Adooq lot validation.

Figure 3A upper panel and caption; associated Results; Methods fluorogenic assays and Proteasome inhibitors

Human LMP2-mediated Ac-PAL-AMC cleavage: Reported fitted IC50 in Results. Figure caption: fluorescence mean ± s.d. from quadruplicate assays; experiments repeated three times with similar results. The ± on fitted IC50 is not separately defined in the Results and is not relabeled here. — Results paragraph linked to Figure 3A; Figure 3A upper panel/caption

LMP7 and LMP2 modification in mouse lymphocytes

Cell-based study · Ficoll-enriched lymphocytes from C57BL/6 mouse spleens [1]

At 300 nM for 2 h, ONX 0914 shifted both LMP7 and LMP2 bands toward higher apparent molecular mass, consistent with covalent modification. This is an in vitro cell exposure, not an animal dosing experiment.

Measured endpoints — not treatment concentrations
Endpoint	Reported result	Reference
Electrophoretic mobility of immunoproteasome subunits	Qualitative immunoblot LMP7 and LMP2 shifted; representative blots from three independent experiments with similar outcome.	[1]

Literature-Reported Treatment Conditions

Treatment concentration: 300 nM
Exposure duration: 2 h
Method: SDS–PAGE and immunoblotting versus DMSO control.
Vehicle: DMSO; shared in vitro final concentration 0.3%.

Methods and source locations

Compound reported in the paper: ONX-0914; Compound label in the paper: ONX 0914 (PR-957)

Applies to named ONX 0914/PR-957 in the stated model; counterion and lot equivalence are not independently established. No salt conversion or Adooq lot validation.

Figure 3B and caption; associated Results; Methods Immunoblotting and Proteasome inhibitors

Electrophoretic mobility of immunoproteasome subunits: Immunoblot includes MECL-1, constitutive catalytic subunits, IOTA and α-tubulin. Band shift is binding evidence, not a direct cellular IC50 or viability measurement. — Figure 3B image/caption and linked Results

Exposure stage affects adipocyte lipid accumulation

Cell-based study · Mouse 3T3-L1 preadipocytes undergoing an eight-day adipocyte differentiation program [2]

ONX 0914 during the first two days reduced final Oil Red O accumulation. Exposure during the following six days produced a nonsignificant tendency toward reduced differentiation. The day-1 viability control does not establish long-term absence of toxicity.

Measured endpoints — not treatment concentrations
Endpoint	Reported result	Reference
Oil Red O accumulation after stage-specific ONX 0914 exposure	Qualitative direction and reported significance Early exposure: reduced accumulation (*p < 0.05); maintenance-phase exposure: reduction tendency not statistically significant.	[2]
Day-1 3T3-L1 viability at tested concentrations	Control assessment Authors report that 1–2 μM ONX0914 was not toxic in the day-1 assay; plotted as % of solvent control.	[2]

Literature-Reported Treatment Conditions

Treatment concentration: 1 or 2 μM
Exposure duration: First 2 days (induction) or following 6 days (maintenance); final lipid endpoint after 8 days.
Method: Oil Red O extraction and eluate absorbance at 492 nm.
Vehicle: DMSO control; final percentage not specified in read Methods.
Co-treatment: Differentiation background: dexamethasone, IBMX and insulin during induction, followed by insulin-containing maintenance medium. ONX 0914 and MG132 were separate treatments.
Reported limits: Exact numerical absorbances are not tabulated and were not digitized; technical replicate count for Figure 3a not specified in caption.

Methods and source locations

Compound reported in the paper: ONX-0914; Compound label in the paper: ONX 0914 (PR-957)

Applies to named ONX 0914/PR-957 in the stated model; counterion and lot equivalence are not independently established. No salt conversion or Adooq lot validation.

Figure 3a and caption; Results “Impaired differentiation of preadipocytes during cultivation of 3T3-L1 cells”; Methods cell culture/differentiation and statistical analysis; Supplemental Figure 2c (PDF page 3)

Oil Red O accumulation after stage-specific ONX 0914 exposure: Figure 3: means ± SD; representative of three independent experiments. Shared statistics: unpaired two-tailed t test. Undifferentiated and DMSO-treated differentiating controls shown. — Figure 3a (both panels)/caption; associated Results

Day-1 3T3-L1 viability at tested concentrations: Trypan-blue cell counting after 1 day. No exact bar values or unverified SI legend statistics extracted; limited duration does not rule out later stress. — Supplemental Figure 2c (PDF page 3); main-text Results and Methods cell-toxicity assay

In Vivo Studies

In a preventive mouse EAE study, ONX-0914 at 10 mg/kg three times weekly from immunization reduced disease incidence and clinical severity in wild-type mice; comparable protection was absent in LMP7-deficient mice. These model-specific results do not establish human efficacy or an exclusively LMP7-mediated effect. [3]

Prophylactic ONX 0914 in autoimmune encephalomyelitis

Animal study · Female C57BL/6 wild-type and LMP7−/− mice with MOG35–55-induced EAE [3]

ONX 0914 reduced disease in wild-type mice when given from the day of immunization. The effect was not reproduced in LMP7-deficient mice. This is prevention in a mouse model, not evidence of clinical efficacy or proof of LMP7-only action.

Measured endpoints — not treatment concentrations
Endpoint	Reported result	Reference
Disease incidence	Count (percentage) Vehicle: 27/30 (90); 10 mg/kg ONX 0914: 6/26 (23.1)	[3]
Day of disease onset	Mean ± s.e.m. Vehicle: 17.2 ± 0.7; ONX 0914: 26.6 ± 1.1 days	[3]
Clinical score at disease peak	Mean ± s.e.m. Vehicle: 2.6 ± 0.1; ONX 0914: 1 ± 0.2	[3]

Conditions shared by the study groups

Frequency: Three times weekly
Treatment course: Beginning on the day of immunization; Table 1 cumulative follow-up ends at day 30.
Vehicle: Aqueous 10% (w/v) sulfobutylether-β-cyclodextrin and 10 mM sodium citrate (pH 6).

Study groups and reported outcomes
Group / dose	Comparator	Observed result	Reference
ONX 0914, wild type 10 mg/kg	Matched vehicle-treated wild type	Lower clinical severity and incidence; delayed onset.	[3]
Vehicle, wild type	ONX 0914-treated wild type	Table 1 incidence 27/30 (90%).	[3]
ONX 0914, LMP7 deficient 10 mg/kg	Vehicle-treated LMP7−/− mice	No comparable protection in Figure 2B.	[3]
Vehicle, LMP7 deficient	ONX 0914-treated LMP7−/− mice	Similar disease course to inhibitor-treated knockout mice.	[3]

ONX 0914, wild type: Model: Female C57BL/6 mice, MOG35–55-induced EAE; Reported limits: Shared Methods allow s.c. or i.v. administration but Figure 2 does not resolve the route for this comparison; route left unspecified.

Vehicle, wild type: Model: Female C57BL/6 mice, MOG35–55-induced EAE; Reported limits: Shared Methods allow s.c. or i.v. administration but Figure 2 does not resolve the route for this comparison; route left unspecified.

ONX 0914, LMP7 deficient: Model: LMP7−/− mice, MOG35–55-induced EAE; Reported limits: Shared Methods allow s.c. or i.v. administration but Figure 2 does not resolve the route for this comparison; route left unspecified.

Vehicle, LMP7 deficient: Model: LMP7−/− mice, MOG35–55-induced EAE; Reported limits: Shared Methods allow s.c. or i.v. administration but Figure 2 does not resolve the route for this comparison; route left unspecified.

Methods and source locations

Compound reported in the paper: ONX-0914; Compound label in the paper: ONX 0914 (PR-957)

Applies to named ONX 0914/PR-957 in the stated model; counterion and lot equivalence are not independently established. No salt conversion or Adooq lot validation.

Figure 2A–B/caption and Table 1/footnotes; associated Results; shared inhibitor, animal and clinical-score/statistics methods

Disease incidence: Cumulative data from five different experiments; the Results prose rounds ONX incidence to 23%. — Table 1 and footnote a

Day of disease onset: Unaffected animals were arbitrarily assigned day 30 for onset (end of experiment); this is not an onset estimate restricted to affected animals. — Table 1 and footnote b

Clinical score at disease peak: Table pools five experiments. Figure 2 shows representative mean clinical scores ± s.e.m. of six mice; 2A performed five times and 2B twice with similar results. No exact curve values inferred. — Table 1; Figure 2A–B/caption

ONX 0914, wild type · Figure 2A–B/caption; Table 1/footnotes; Methods Proteasome inhibition in mice

Vehicle, wild type · Figure 2A–B/caption; Table 1/footnotes; Methods Proteasome inhibition in mice

ONX 0914, LMP7 deficient · Figure 2A–B/caption; Table 1/footnotes; Methods Proteasome inhibition in mice

Vehicle, LMP7 deficient · Figure 2A–B/caption; Table 1/footnotes; Methods Proteasome inhibition in mice

Specifications

Manufacturer	Adooq Bioscience
Actions	Inhibitor
CAS Number	960374-59-8
Applications	Immunoproteasome inhibitor
Formula	C₃₁H₄₀N₄O₇
Molecular Weight	580.67
Synonyms	PR-957
SMILES	C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)OC)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)[C@]3(CO3)C)NC(=O)CN4CCOCC4

Storage & Solubility

In Vitro Solubility

Reported Solubility

Solvent	Reported Solubility
DMSO	87 mg/mL (149.82 mM)
Water	Insoluble
Ethanol	87 mg/mL (149.82 mM)

Preparing Stock Solutions

Stock preparation is unavailable for this product molecular weight.

Calculated stock concentrations do not establish solvent solubility. Refer to the reported solubility data for experimental guidance.

In Vivo Preparation

Concentration: 9 mg/mL
Vehicle: 2% DMSO+castor oil

Storage

Store powder at -20°C, keep desiccated.
In powder form, the chemical is stable for 36 months.
In solution, store at -20°C and use within 1 month to prevent loss of potency. Aliquot to avoid multiple freeze/thaw cycles.

Research References

[1] Basler M, Lindstrom MM, LaStant JJ, et al. Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity EMBO reports (2018). PMID: 30279279 DOI: 10.15252/embr.201846512 Source material
[2] Arimochi H, Sasaki Y, Kitamura A, et al. Differentiation of preadipocytes and mature adipocytes requires PSMB8 Scientific reports (2016). PMID: 27225296 DOI: 10.1038/srep26791 Source material
[3] Basler M, Mundt S, Muchamuel T, et al. Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis EMBO molecular medicine (2014). PMID: 24399752 DOI: 10.1002/emmm.201303543 Source material

Publications

5 publications citing this product

David J Zahavi, .et al. , Cancer Biol Ther, 2023, Dec 31;24(1):2269637 PMID: 37878417
Yuki Sasaki, .et al. , JCI Insight, 2022, Apr 8;7(7) PMID: 35393946
Ming-Shu Mo, .et al. , Transl Neurodegener, 2018, 7: 19 PMID: 30128145
Ohkusu-Tsukada K, .et al. , Int Arch Allergy Immunol, 2018, 176(2):91-100 PMID: 29669333
Arimochi H, .et al. , Sci Rep, 2016, May 26;6:26791 PMID: 27225296

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ONX-0914

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Biological Activity

Bioactivity / Mechanism

Target & Activity

Cellular Effect

In Vitro Studies

Human immunoproteasome LMP2 inhibition

Literature-Reported Treatment Conditions

LMP7 and LMP2 modification in mouse lymphocytes

Literature-Reported Treatment Conditions

Exposure stage affects adipocyte lipid accumulation

Literature-Reported Treatment Conditions

In Vivo Studies

Prophylactic ONX 0914 in autoimmune encephalomyelitis

Conditions shared by the study groups

Specifications

Storage & Solubility

In Vitro Solubility

Reported Solubility

Preparing Stock Solutions

In Vivo Preparation

Storage

Research References

Publications

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