APH003
APH003 is an orally active IRAK4 PROTAC degrader with a DC50 of 0.74 nM in human peripheral blood mononuclear cells (hPBMCs). APH003 recruits IRAK4 to CRBN to form a ternary complex, mediates the ubiquitination and degradation of IRAK4 via the ubiquitin-proteasome pathway, and inhibits the kinase activity of IRAK4. APH003 inhibits LPS- or IL-1β/LPS-induced phosphorylation of ERK, JNK and NF-κB. APH003 inhibits the secretion of TNF-α, IL-6, IL-8 and IL-13 by stimulated hPBMCs. APH003 exhibits anti-inflammatory activity in rat TNBS-induced intestinal inflammation models and mouse IL-33-induced skin inflammation models. APH003 can be used in research related to inflammatory bowel disease and skin inflammation.
(Pink: IRAK4 ligand (HY-184916); Blue: Cereblon ligand (HY-49385); Black: linker).
For research use only. We do not sell to patients.
- CAS No.: 3065495-08-8
- Formula: C44H50FN11O7
- Molecular Weight:863.94
-
Storage:
Please store the product under the recommended conditions in the Certificate of Analysis.
To place orders, for customer services and technical support, please contact: MedChemExpress USA
Tel: 609-228-6898 E-mail: [email protected] [email protected]
-
Biological Activity
-
Chemical Information
- Protocols
- Purity & Documentation
- References
-
Help & FAQs
All PROTACs Isoforms
More
Biological Activity
Description
IC50 & Target
[1]|
IRAK4 1.13 nM (IC50) |
IRAK4 4.76 nM (EC50) |
IRAK4 75.26 nM (Kd) |
ERK |
NF-κB |
JNK |
IL-1β |
IL-6 |
IL-8 |
IL-13 |
In Vitro
APH003 (10-point concentration gradient; 60 min) potently inhibits purified IRAK4 kinase, with an IC50 of 1.13 nM[1].
APH003 (0.001-10000 nM; 15 min) binds to purified CRBN with a Kd value of 75.26 nM[1].
APH003 (0.001-1000 nM; 60 min) promotes the formation of the IRAK4-PROTAC-CRBN ternary complex, with an EC50 of 4.76 nM[1].
APH003 (0.05-1000 nM; 2-48 h) degrades IRAK4 in human peripheral blood mononuclear cells (hPBMCs) via the CRBN-ubiquitin-proteasome pathway, with a 24 h DC50 of 0.74 nM, and its potency increases in a time-dependent manner within 48 h[1].
APH003 (0.05-1000 nM; 24 h) degrades IRAK4 in mouse, rat and canine PBMCs, with corresponding DC50 values of 5.06 nM, 2.24 nM and 3.88 nM, respectively, and its potency is lower than that in human PBMCs (hPBMCs)[1].
APH003 (0.05-1000 nM; 24 h pretreatment, 30 min LPS stimulation) inhibits LPS-induced phosphorylation of ERK, JNK, and NF-κB in human peripheral blood mononuclear cells (hPBMCs), with IC50 values of 31.1 nM, 206.5 nM, and 353.8 nM, respectively[1].
APH003 (0.03-2000 nM; 24 h pretreatment, 24 h LPS stimulation) inhibits LPS-induced secretion of TNF-α and IL-6 in human peripheral blood mononuclear cells (hPBMCs), with IC50 values of 3.84 nM and 2.86 nM, respectively[1].
APH003 (0.03-2000 nM; 24 h pretreatment, 24 h costimulation) inhibits the secretion of TNF-α, IL-6, IL-8 and IL-13 in human peripheral blood mononuclear cells (hPBMCs) stimulated by IL-1β + LPS, with corresponding IC50 values of 12.06 nM, 17.34 nM, 112.92 nM and 43.27 nM, respectively[1].
APH003 (10-1000 nM; 24 h) selectively degrades IRAK4 in human peripheral blood mononuclear cells (hPBMCs), and does not affect IRAK family isoforms or novel CRBN substrates even at a concentration of 1000 nM[1].
APH003 (60 nM; 24 h) exhibits highly selective degradation of IRAK4 in human peripheral blood mononuclear cells (hPBMCs), with minimal effects on other cellular proteins[1].
APH003 exhibits excellent metabolic stability (T1/2 > 371 min) in human, monkey and dog hepatocytes, and moderate stability in rat and mouse hepatocytes[1].
APH003 exhibits weak inhibitory effects on human CYP subtypes, indicating a low potential for drug-drug interactions mediated by CYP inhibition[1].
APH003 exhibits moderate Caco-2 permeability with an efflux ratio of 7.36, suggesting that it may be a substrate of efflux transporters[1].
APH003 shows low solubility in neutral/weakly acidic media but high solubility in acidic FaSSGF, which supports its potential oral absorbability[1].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only. Further protocols information, click here.
-
Cell Line:Human PBMCs
-
Concentration:0.05 nM, 0.15 nM, 0.46 nM, 1.37 nM, 4.12 nM, 12.35 nM, 37.04 nM, 111.11 nM, 333.33 nM, 1000.00 nM
-
Incubation Time:24 h
-
Result:Inhibited LPS-induced phosphorylation of ERK, JNK, and NF-κB in human peripheral blood mononuclear cells (hPBMCs).
-
Cell Line:Human PBMCs
-
Concentration:10 nM, 100 nM, 100 nM
-
Incubation Time:24 h
-
Result:Selectively degraded IRAK4 in human peripheral blood mononuclear cells (hPBMCs).
Did not affect IRAK family isoforms or novel CRBN substrates even at a concentration of 1000 nM.
Parmacokinetics
| Species | Dose | Route | Cmax | AUClast | T1/2 | CL | Vss | Tmax | F |
|---|---|---|---|---|---|---|---|---|---|
| Mice[1] | 2 mg/kg | i.v. | 3270 ng/mL | 12939 ng·h/mL | 4.68 h | 1.88 mL/min/kg | 0.749 L/kg | / | / |
| Mice[1] | 10 mg/kg | i.g. | 4830 ng/mL | 38237 ng·h/mL | 11.3 h | / | / | 1.00 h | 68 % |
| Rat[1] | 2 mg/kg | i.v. | 1245 ng/mL | 4500 ng·h/mL | 1.46 h | 7.40 mL/min/kg | 0.729 L/kg | / | / |
| Rat[1] | 30 mg/kg | i.g. | 3563 ng/mL | 31463 ng·h/mL | 2.78 h | / | / | 2.00 h | 46 % |
| Dog[1] | 1 mg/kg | i.v. | 548 ng/mL | 3367 ng·h/mL | 12.2 h | 4.69 mL/min/kg | 3.84 L/kg | / | / |
| Dog[1] | 5 mg/kg | i.g. | 491 ng/mL | 10119 ng·h/mL | 14.4 h | / | / | 4.00 h | 63 % |
| Cynomolgus Monkey[1] | 1 mg/kg | i.v. | 680 ng/mL | 2978 ng·h/mL | 10.5 h | 5.43 mL/min/kg | 3.68 L/kg | / | / |
| Cynomolgus Monkey[1] | 20 mg/kg | i.g. | 1423 ng/mL | 18843 ng·h/mL | 11.3 h | / | / | 4.00 h | 32 % |
In Vivo
APH003 (3-30 mg/kg; p.o.; twice daily; 11 days) dose-dependently suppresses IL-33-induced skin inflammation in mice, with a 68% inhibition of delta ear thickness AUC0-t and 97% inhibition of ear tissue IL-5 secretion at the 30 mg/kg twice-daily oral dose, accompanied by IRAK4 degradation in circulating WBCs[1].
APH003 (3-100 mg/kg; p.o.; daily; 7 days) dose-dependently degrades IRAK4 in rat skin and PBMCs after repeated oral administration, achieving 91% IRAK4 degradation in skin at the 100 mg/kg daily dose[1].
APH003 (0.3-3 mg/kg; p.o.; daily; 7 days) degrades IRAK4 in dog PBMCs and skin after repeated oral administration, achieving 88% IRAK4 degradation in skin at the 3 mg/kg daily dose after 7 days, with skin degradation showing no consistent dose-dependent trend across the tested doses[1].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
-
Animal Model:Sprague-Dawley (SD) rats (male)[1]
-
Dosage:10 mg/kg; 30 mg/kg; 100 mg/kg
-
Administration:p.o.; daily; 8 days
-
Result:Significantly alleviated TNBS-induced weight loss on day 8, reduced DAI scores starting on day 6, and inhibited the AUC of DAI scores by 37% at 10 mg/kg.
Reduced DAI scores starting on day 3, and inhibited the AUC of DAI scores by 56% at 30 mg/kg.
Significantly alleviated weight loss from day 5 to day 8, reduced DAI scores starting on day 3, and inhibited the AUC of DAI scores by 64% at 100 mg/kg.
Degraded IRAK4 in rat colon tissue by 94% at 10 mg/kg, 92% at 30 mg/kg, and 94% at 100 mg/kg.
-
Animal Model:male[1]
-
Dosage:3 mg/kg; 10 mg/kg; 30 mg/kg
-
Administration:p.o.; twice daily; 11 days
-
Result:Reduced delta ear thickness in a dose-dependent manner; at 30 mg/kg, almost completely eliminated IL-33-induced ear inflammation by day 11.
Inhibited the AUC of delta ear thickness by 52% at 3 mg/kg, 55% at 10 mg/kg, and 68% at 30 mg/kg.
Inhibited IL-5 secretion in ear tissue by 59% at 3 mg/kg, 55% at 10 mg/kg, and 97% at 30 mg/kg; in plasma, inhibited IL-5 secretion by 55% at 3 mg/kg, 61% at 10 mg/kg, and 96% at 30 mg/kg.
Degraded IRAK4 in WBCs by 53% at 3 mg/kg, 68% at 10 mg/kg, and 71% at 30 mg/kg.
-
Animal Model:Sprague-Dawley (SD) rats (male)[1]
-
Dosage:3 mg/kg; 100 mg/kg
-
Administration:p.o.; daily; 7 days
-
Result:Degraded IRAK4 in rat skin by 55% 24 hours after the seventh dose; in PBMCs, degraded IRAK4 by 78% at 6 hours and 75% at 24 hours after the first dose, and by 76% at 6 hours and 71% at 24 hours after the seventh dose at 3 mg/kg.
Degraded IRAK4 in rat skin by 91% 24 hours after the seventh dose; in PBMCs, degraded IRAK4 by 92% at 6 hours and 91% at 24 hours after the seventh dose at 100 mg/kg.
-
Animal Model:Beagle dogs (male)[1]
-
Dosage:0.3 mg/kg; 1 mg/kg; 3 mg/kg
-
Administration:p.o.; daily; 7 days
-
Result:Degraded IRAK4 in dog PBMCs by 78% 24 hours after the first dose and 60% 24 hours after the seventh dose; in skin, degraded IRAK4 by 41% 24 hours after the first dose and 37% 24 hours after the seventh dose at 0.3 mg/kg.
Degraded IRAK4 in dog PBMCs by 82% 24 hours after the first dose and 72% 24 hours after the seventh dose; in skin, degraded IRAK4 by 41% 24 hours after the first dose and 22% 24 hours after the seventh dose at 1 mg/kg.
Degraded IRAK4 in dog PBMCs by 82% 24 hours after the first dose and 83% 24 hours after the seventh dose; in skin, degraded IRAK4 by 31% 24 hours after the first dose and 88% 24 hours after the seventh dose at 3 mg/kg.
Chemical Information
-
CAS No. 3065495-08-8
-
Molecular Weight 863.94
-
Formula C44H50FN11O7
-
SMILES
FC1=C(C=C2N(N=C(C2=C1)N3C(NC(CC3)=O)=O)C)N4CCC(O)(CC4)CC(N5CCC6(CC5)CC7=C(O6)C=C(N8CCC(CC8)CO)C(NC(C9=C%10N=CC=CN%10N=C9)=O)=C7)=O
-
Shipping
Room temperature in continental US; may vary elsewhere.
-
Storage
Please store the product under the recommended conditions in the Certificate of Analysis.
Protocols
-
Western Blot
Western blotting (WB) is a commonly used experimental method in molecular biology, biochemistry, and immunogenetics for identifying and quantifying target proteins. It combines gel electrophoresis with immunoassay, enabling researchers to analyze protein expression, post-translational modifications, and molecular weight.
-
Research Protocol for Inflammation-related Diseases
The NLRP3 inflammasome is a cytosolic innate immune signaling platform that integrates priming signals and danger-signal activation to promote caspase-1 activation, maturation of IL-1β and IL-18, and gasdermin D-mediated pyroptotic cell death. The core experimental logic is to determine whether inflammatory disease phenotypes are driven by increased NLRP3 expression, ASC-containing inflammasome assembly, caspase-1 cleavage, GSDMD cleavage, and extracellular release of IL-1β/IL-18 rather than by nonspecific cell injury alone. The pathway is strongly linked to inflammation-related disease phenotypes because monosodium urate crystals activate NALP3/NLRP3 inflammasome signaling in gout-like crystal inflammation, cholesterol crystals activate NLRP3 inflammasomes in atherogenesis models, and DSS-induced intestinal inflammation has been reported to involve NLRP3 inflammasome activity. However, experimental colitis studies also show context-dependent protective effects of NLRP3 inflammasome co
-
Kinase activity and phosphorylation assays
Kinase activity assays measure the ability of kinases to transfer phosphate groups from ATP to specific substrates, while phosphorylation assays detect the presence and levels of phosphorylated proteins. Common methods include radiolabeled ATP incorporation (e. g. ,), ADP release detection via bioluminescence (e. g. ,[3]), enzyme-linked immunosorbent assays (ELISA) for phospho-specific epitopes (e. g. ,[6]), and microtiter-based formats for high-throughput screening (e. g. ,[8]). The ADP-Glo assay quantifies kinase activity by measuring ADP produced during phosphorylation using a luciferase-based system. Radiometric assays involve autoradiography or scintillation counting after incorporation of 32P-labeled ATP into substrate proteins. ELISA-based approaches rely on phospho-specific antibodies to detect activated kinases in cell lysates or purified samples.
-
Imiquimod-Induced Psoriasiform Dermatitis
Imiquimod (IMQ)-induced psoriasiform dermatitis is a widely used murine model in which topical application of IMQ, a Toll-like receptor 7 (TLR7) agonist, triggers innate immune activation in the skin and induces a psoriasis-like inflammatory cascade characterized by epidermal hyperplasia, immune cell infiltration, and cytokine production dominated by the IL-23/IL-17 axis. This inflammatory response is mediated through activation of dendritic cells and downstream induction of IL-23, IL-17A, IL-22, and related pro-inflammatory mediators, recapitulating key features of human plaque psoriasis and enabling mechanistic and therapeutic studies. The model is commonly induced using Aldara (5% IMQ cream) applied topically to murine skin, resulting in rapid onset of erythema, scaling, and thickening that can be quantified as disease severity indices and validated histologically.
-
PBMC Thawing for Immune Assays
PBMC thawing for immune assays recovers viable cryopreserved peripheral blood mononuclear cells for downstream functional or phenotypic readouts, including ELISPOT, intracellular cytokine staining, proliferation assays, and flow-cytometric immunophenotyping. Cryopreserved PBMCs can support immune monitoring because antigen-specific T-cell function and major CD4/CD8 phenotypes may be retained after optimized freezing and thawing, although some lymphocyte subsets and activation or memory markers can be altered by cryopreservation. The technical objective is rapid warming of the frozen vial followed by controlled dilution and removal of DMSO-containing cryomedium, because thawing and wash conditions measurably affect viable PBMC recovery and downstream assay performance. Viability alone is insufficient for protocol evaluation because high viability may occur with low live-cell recovery, so both viable percentage and absolute live-cell recovery should be measured after thawing.
-
Cotton Pellet Granuloma
Cotton pellet granuloma is a classical in vivo chronic inflammation model used to evaluate the anti-inflammatory potential of test substances by measuring their ability to inhibit granuloma tissue formation around an implanted foreign body (cotton pellet) in rodents. The method is based on the biological response to a sterile implanted material, which induces proliferative phase inflammation characterized by fibroblast proliferation and collagen-rich granuloma formation, and the final readout reflects the extent of chronic inflammatory tissue growth surrounding the pellet. In multiple preclinical pharmacological evaluations, inhibition of cotton pellet-induced granuloma formation has been used as an indicator of anti-inflammatory activity in both synthetic and natural product screening contexts.
-
Carrageenan-Induced Paw Edema
Carrageenan-induced paw edema is an acute inflammation model in which intraplantar injection of carrageenan induces localized inflammatory swelling characterized by vascular permeability, leukocyte infiltration, and production of inflammatory mediators such as prostaglandins and cytokines, making it widely used to evaluate anti-inflammatory agents in vivo. The resulting paw volume or thickness increase is quantified over time as a direct readout of inflammatory intensity and drug efficacy, typically reflecting cyclooxygenase-mediated prostaglandin-driven edema formation and immune cell recruitment in peripheral tissue[20].
-
DSS-Induced Colitis
Dextran sulfate sodium (DSS)-induced colitis is generated by administering DSS in mouse drinking water, producing epithelial injury, barrier disruption, weight loss, diarrhea, fecal blood, colon shortening, histologic mucosal damage, and inflammatory mediator changes; the model is mainly used to study acute or chronic intestinal inflammation resembling selected features of ulcerative colitis. DSS injury is interpreted through clinical and tissue readouts rather than a single molecular endpoint: daily body weight, stool consistency, and bleeding are combined into a disease activity index, while colon length, histology, cytokines, myeloperoxidase activity, intestinal permeability, and tight-junction markers provide complementary measures of inflammation and barrier damage.
-
TNBS-Induced Colitis
TNBS-induced colitis is produced by intrarectal delivery of 2,4,6-trinitrobenzene sulfonic acid in ethanol, where ethanol disrupts the mucosal barrier and TNBS haptenates colonic proteins, generating immune-mediated colonic inflammation with weight loss, diarrhea, ulceration, transmural injury, inflammatory-cell infiltration, and cytokine responses. The model is used as an experimental intestinal inflammation model with Crohn’s disease–like features, especially when Th1-type responses, IL-12–dependent inflammation, chronic relapsing inflammation, or fibrosis-related endpoints are studied.
-
TPA/Croton Oil Ear Edema and Dermatitis
The TPA (12-O-tetradecanoylphorbol-13-acetate) and croton oil-induced mouse ear edema model is a well-established acute cutaneous inflammation system used to evaluate topical anti-inflammatory activity by measuring edema formation, neutrophil infiltration, vascular permeability, and cytokine-mediated skin responses in vivo. The inflammatory response is triggered by topical application of phorbol esters (TPA) or croton oil constituents, leading to rapid activation of protein kinase C signaling, leukocyte recruitment, and increased vascular permeability, which can be quantified by ear thickness, weight, dye extravasation, and biochemical markers such as myeloperoxidase (MPO) activity and pro-inflammatory mediators in ear tissue homogenates. This model is widely used for screening anti-inflammatory agents, where reductions in edema and inflammatory biomarkers reflect suppression of acute dermal inflammation and immune cell infiltration. Histological evaluation typically confirms epidermal
-
Contact Hypersensitivity Dermatitis
Contact hypersensitivity (CHS) dermatitis is a T cell-mediated delayed-type (Type IV) immune reaction in which low-molecular-weight haptens applied to the skin bind host proteins to form complete antigens, triggering sensitization followed by a secondary inflammatory response upon re-exposure (elicitation phase), which is commonly quantified by ear swelling as a readout of skin inflammation in murine models. This model is widely used to study allergic contact dermatitis because it is antigen-specific, reproducible, and reflects key immunological events including dendritic cell activation, T cell priming in draining lymph nodes, and effector T cell-driven tissue inflammation. DNFB- and oxazolone-induced CHS models are standard systems for evaluating both acute and chronic T cell-dependent skin inflammation and for testing immunomodulatory interventions.
-
Protocol for Kinase activity and phosphorylation assays
Kinase activity assays measure transfer of phosphate from ATP to a protein or peptide substrate, generating phosphorylated substrate, ADP, or incorporated radiolabeled phosphate as the readout; phosphorylation assays measure site-specific phosphorylation in cells or tissues as a proxy for kinase-pathway activation, inhibition, or substrate regulation. Phosphorylation can be detected by phospho-specific Western blot, immunoprecipitation kinase assay, phospho-immunofluorescence, phospho-flow cytometry, luminescent ADP detection, radiolabeled ATP incorporation, or reporter-based pathway assays, and these readouts can be applied to cancer cells, primary neurons, mouse tumors, organoids, inflammatory macrophages, ferroptosis studies, and mitophagy studies when the kinase target is biologically relevant.
-
Pyroptosis Solutions
Pyroptosis is a lytic inflammatory cell-death pathway executed by gasdermin pores, most classically through inflammasome-mediated activation of caspase-1, cleavage of gasdermin D, membrane pore formation, LDH release, and secretion of IL-1β and IL-18. The canonical pathway is commonly modeled by priming cells with an inflammatory signal such as LPS to induce pro-IL-1β and inflammasome components, followed by an activation signal such as ATP or nigericin to activate NLRP3, ASC speck formation, caspase-1 cleavage, GSDMD cleavage, cytokine release, and pyroptotic membrane rupture. The non-canonical pathway is triggered when cytosolic LPS activates mouse caspase-11 or human caspase-4/5, leading to GSDMD cleavage and pyroptosis, and this can secondarily activate NLRP3-dependent IL-1β release. Pyroptosis is linked to inflammatory injury, infection, cancer, liver disease, ocular disease, placental inflammation, and other disease phenotypes, but unresolved questions include which gasdermin fam
Purity & Documentation
References
Calculators
Concentration (start) × Volume (start) = Concentration (final) × Volume (final)
APH003 Related Classifications
HY-184915 Related Classifications
Powered by Bioz