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| Targets |
Bruton's tyrosine kinase (BTK) ; DC50 = 0.34 nM; CRBN
The target of Bexobrutideg is Bruton's tyrosine kinase (BTK). As a PROTAC, it consists of two functional moieties: a BTK-binding ligand and a cereblon E3 ubiquitin ligase-binding ligand, connected by a linker. The compound brings BTK into proximity with cereblon, leading to ubiquitination and subsequent proteasomal degradation of BTK. Unlike traditional inhibitors, degrading BTK eliminates both its enzymatic and scaffolding functions, and it does not degrade other cereblon neo-substrates such as the transcription factors Aiolos and Ikaros, thus lacking immunomodulatory activity. Bexobrutideg targets BTK, a key kinase involved in B-cell receptor (BCR) signaling. Unlike conventional inhibitors, it is a heterobifunctional molecule that recruits an E3 ubiquitin ligase (cereblon) to BTK, leading to its ubiquitination and subsequent degradation by the proteasome. This targeted degradation results in the sustained inhibition of BTK signaling, which is essential for the survival and proliferation of B cells. |
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| ln Vitro |
- NX-5948 is a potent degrader of BTK in primary human B cells with a DC50 of 0.34 nM. It also inhibits B cell receptor (BCR) signaling. [1]
- Proteomic analysis shows NX-5948 is highly selective for BTK degradation, with limited activity toward the CRBN neo-substrate Aiolos (DC50 > 10 µM). [1] - NX-5948 induces degradation of wild-type and mutant forms of BTK in B-cells at sub-nanomolar potencies. [2] In vitro studies demonstrate that Bexobrutideg is a highly potent BTK degrader with remarkable catalytic efficiency. At clinically relevant concentrations, a single molecule can degrade approximately 10,000 copies of BTK protein per hour. The compound induces specific BTK protein degradation via the cereblon E3 ligase complex, leading to inhibition of B cell activation and potent anti-inflammatory activity. Bexobrutideg exhibits potent tumor growth inhibition in TMD8 xenograft models containing either wild-type BTK or BTKi-resistant mutations such as C481S. In vitro, Bexobrutideg has been shown to selectively degrade BTK in B-cell lines. By degrading BTK, it inhibits BCR signaling and induces apoptosis in malignant B cells. Its activity is assessed using cellular assays that measure BTK protein levels and downstream signaling pathways. These in vitro studies confirm its potential as a therapeutic agent for B-cell malignancies. |
| ln Vivo |
- In vivo, once daily oral administration of NX-5948 in mice and cynomolgus monkeys demonstrated potent degradation of BTK in circulating B cells. [1]
- NX-5948 demonstrated significant anti-inflammatory activity and resulted in improvement of clinical symptoms in a mouse collagen-induced arthritis (CIA) model. [1] - NX-5948 exhibits potent tumor growth inhibition in TMD8 xenograft models that contain either wild-type BTK or BTK-resistant mutations. [2] Bexobrutideg demonstrates activity in multiple in vivo models. In a mouse collagen-induced arthritis (CIA) model, the compound effectively alleviates inflammatory symptoms. More importantly, in Phase 1a/b clinical trials, Bexobrutideg shows high clinical activity in patients with relapsed/refractory B-cell malignancies. As of May 2025 data, the objective response rate (ORR) in Waldenström macroglobulinemia patients was 85.0%, with 3 patients achieving very good partial responses. In chronic lymphocytic leukemia patients, the ORR was 80.9%. Robust, rapid, and sustained BTK degradation was observed at all dose levels, with steady declines in IgM levels and deepening responses over time. In vivo, Bexobrutideg has demonstrated anti-tumor activity in animal models of B-cell malignancies. Its ability to penetrate the blood-brain barrier allows it to target BTK in the central nervous system, which is relevant for diseases like primary central nervous system lymphoma. These in vivo studies provide evidence for its therapeutic potential. |
| Enzyme Assay |
As a PROTAC, the activity of Bexobrutideg depends on an intact intracellular ubiquitin-proteasome system, rendering classic enzyme/receptor binding assays less directly applicable. Its targeted degradation activity is typically assessed in cell-based models. In cell-free systems, surface plasmon resonance (SPR) can be utilized to evaluate its binding affinity to BTK or the E3 ligase cereblon. Recombinant BTK or cereblon protein is immobilized on a sensor chip, and varying concentrations of Bexobrutideg (0.1 nM-10 μM) are flowed over to determine binding kinetics and affinity (Kd). However, functional (degradation) activity must be validated in cellular assays.
Cell-free assays for Bexobrutideg involve measuring its ability to induce BTK degradation in a cell-free system. These assays typically use purified components to reconstitute the ubiquitination and degradation process. The compound's potency is assessed by measuring the extent of BTK degradation in the presence of varying concentrations of the compound. |
| Cell Assay |
- BTK Degradation Assay: Robust degradation of BTK was observed by flow cytometry in primary human B cells after 4-hour treatment with NX-5948. The half-maximal degradation concentration (DC50) was determined to be 0.34 nM. [1]
- Proteomic Analysis: The selectivity of NX-5948 for BTK degradation was assessed via proteomic analysis, which demonstrated minimal off-target degradation, particularly showing a DC50 > 10 µM for the CRBN neo-substrate Aiolos. [1] - Assessment of BTK Degradation in Patients: In a Phase 1a trial, BTK expression in patients' blood samples was measured to evaluate degradation. Rapid, robust, and sustained BTK degradation was observed across all patients, irrespective of baseline BTK level, tumor type, or NX-5948 dose. [2] A standard in vitro cell assay protocol for Bexobrutideg is as follows: 1) Seed target cells (such as TMD8, Ramos B-cell lines, or primary CLL cells) in culture plates and culture at 37°C with 5% CO₂; 2) Treat cells with a range of concentrations of Bexobrutideg (e.g., 0.01 nM-1 μM) for 4-24 hours; 3) Harvest cell lysates and measure BTK protein levels by Western blot using GAPDH or Vinculin as loading controls to calculate the DC₅₀ (half-maximal degradation concentration); 4) Assess changes in B-cell activation markers (e.g., CD69, CD86) by flow cytometry; 5) Measure cell viability using CellTiter-Glo or CCK-8 assays; 6) Pre-incubate with a proteasome inhibitor (e.g., MG132) when necessary to verify that degradation is mediated via the proteasome pathway. In vitro cellular assays are conducted to evaluate the functional activity of Bexobrutideg. B-cell lines are treated with the compound, and BTK protein levels are measured using Western blotting to confirm degradation. BCR signaling is assessed by measuring the phosphorylation of downstream targets. Cell proliferation and apoptosis assays are used to evaluate the compound's anti-tumor activity. These assays confirm that Bexobrutideg effectively degrades BTK and inhibits BCR signaling. |
| Animal Protocol |
- Mouse Collagen-Induced Arthritis (CIA) Model: The anti-inflammatory activity of NX-5948 was evaluated in a mouse CIA model. The compound was administered orally once daily. The study showed significant anti-inflammatory activity and improvement in clinical symptoms. [1]
- In Vivo Pharmacodynamic Study in Mice and Cynomolgus Monkeys: To assess BTK degradation in vivo, NX-5948 was administered orally once daily to mice and cynomolgus monkeys. Potent degradation of BTK in circulating B cells was subsequently observed. [1] - TMD8 Xenograft Model: The anti-tumor efficacy of NX-5948 was evaluated in TMD8 xenograft models, which include both wild-type BTK and BTK-resistant mutations. The study demonstrated potent tumor growth inhibition. [2] An in vivo animal assay protocol for Bexobrutideg is as follows: 1) Use immunodeficient mice (such as NSG or SCID mice); 2) Establish xenograft or systemic leukemia models by subcutaneous or intravenous injection of tumor cells (e.g., TMD8-luc or other B-cell lymphoma cells); 3) When tumors reach approximately 100-300 mm³, randomize animals into groups (8-10 per group), including vehicle control and Bexobrutideg treatment groups (doses typically 10, 30, 100 mg/kg); 4) Administer by oral gavage once or twice daily for 2-4 weeks; 5) Measure tumor volume and body weight 2-3 times weekly; 6) At study termination, euthanize animals and collect tumor tissue, blood, and major organs for BTK degradation assessment by Western blot and histopathological examination. Additionally, the compound has demonstrated anti-inflammatory activity in a mouse collagen-induced arthritis (CIA) model. In vivo animal experiments typically involve xenograft models of B-cell malignancies. Animals are administered the compound via oral gavage. Tumor growth is monitored over time to assess efficacy. Pharmacokinetic studies are conducted to measure plasma and brain concentrations of the compound. |
| ADME/Pharmacokinetics |
- In Humans (Phase 1a Trial): NX-5948 exhibits dose-dependent pharmacokinetics (PK) with a half-life of approximately 24 hours, supporting once-daily dosing. [2]
Bexobrutideg is an orally bioavailable small molecule degrader with favorable pharmacokinetic properties. Ongoing Phase 1 clinical trials are evaluating its pharmacokinetic profile in humans, including blood concentration, distribution, metabolism, and excretion characteristics. Preclinical studies show that the compound can cross the blood-brain barrier, as it has been detected in the cerebrospinal fluid of preclinical models and has demonstrated clinical efficacy in patients with primary CNS lymphoma or CLL with CNS involvement. Its catalytic degradation mechanism suggests that efficient degradation and clinical activity can be achieved at relatively low drug concentrations. The pharmacokinetic properties of Bexobrutideg are characteristic of an orally active small molecule PROTAC. It has a molecular weight of 806.96. The compound is designed to have favorable drug-like properties, including good oral bioavailability and blood-brain barrier penetration. Pharmacokinetic studies in animal models involve measuring plasma and brain concentrations of the compound over time to determine its half-life, clearance, and volume of distribution. |
| Toxicity/Toxicokinetics |
- In Humans (Phase 1a Trial): NX-5948 was well tolerated with no dose-limiting toxicities (DLTs). There were no treatment-emergent adverse events (TEAEs) resulting in drug discontinuation or dose reduction, and no drug-related Grade ≥3 TEAEs or related serious adverse events. The most common TEAEs were purpura/contusion (57.1%, all below grade 3), nausea (35.7%), and thrombocytopenia (35.7%). No atrial fibrillation/flutter or hypertension was reported. [2]
Based on published Phase 1 clinical trial data, Bexobrutideg is well-tolerated with a manageable safety profile across all dose levels. The most common treatment-emergent adverse events include diarrhea, thrombocytopenia, purpura/contusion, petechiae, and neutropenia, mostly Grade 1-2. No dose-limiting toxicities were observed, and no clinically significant atrial or ventricular arrhythmias have been reported. No systemic fungal infections were reported, and treatment discontinuation rates were low. Unlike traditional BTK inhibitors, this degrader does not target IKZF1 and IKZF3 for degradation and thus lacks immunomodulatory activity, which may contribute to its favorable safety profile. The toxicity profile of Bexobrutideg is not extensively documented, but it is likely to be similar to other BTK inhibitors. Common adverse effects may include diarrhea, fatigue, and infections. As a PROTAC, it may have a different safety profile compared to traditional inhibitors. Preclinical toxicology studies would typically involve acute and chronic dosing in animal models to assess safety margins. |
| References | |
| Additional Infomation |
- Background & Mechanism: NX-5948 is a chimeric targeting molecule (CTM) that engages the E3 ligase cereblon (CRBN) to promote the selective degradation of BTK. It is an orally administered small molecule. [1]
- Clinical Development: Preclinical animal models support the clinical development of NX-5948 for treating autoimmune diseases. [1] A Phase 1a/b first-in-human trial (NX-5948-301) is being conducted in patients with relapsed/refractory B cell malignancies, including chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL). [2] - Clinical Activity (Phase 1a): In heavily pre-treated patients, early signs of clinical activity were observed. Of three evaluable CLL patients receiving the lowest dose of 50 mg, one confirmed partial response (PR) and two patients had stable disease (SD) at the 8-week assessment. [2] Bexobrutideg is being investigated in clinical trials for the treatment of B-cell malignancies, including B-cell lymphoma, chronic lymphocytic leukemia (CLL), and small lymphocytic lymphoma (SLL). It is currently in Phase 3 clinical trials. Its mechanism of action involves the targeted degradation of BTK, offering a potential advantage over traditional BTK inhibitors by providing sustained target inhibition. |
| Molecular Formula |
C42H54N12O5
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|---|---|
| Molecular Weight |
806.955567836761
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| Exact Mass |
806.434
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| Elemental Analysis |
C, 62.51; H, 6.75; N, 20.83; O, 9.91
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| CAS # |
2649400-34-8
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| Related CAS # |
2649400-33-7
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| PubChem CID |
156464216
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| Appearance |
Off-white to yellow solid powder
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| LogP |
2.6
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
12
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
59
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| Complexity |
1500
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| Defined Atom Stereocenter Count |
2
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| SMILES |
C1(C(N)=O)=NC=C(N2CCC[C@@H](N3C(=O)N(C)CC3)C2)N=C1NC1=CC=C(C2CCN(CC3CCN(C4=CC=C(C(N[C@H]5CCC(=O)NC5=O)=O)N=C4)CC3)CC2)C=C1
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| InChi Key |
HPTPDBYCFHFWJG-CWTKIQHKSA-N
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| InChi Code |
InChI=1S/C42H54N12O5/c1-50-21-22-54(42(50)59)32-3-2-16-53(26-32)35-24-45-37(38(43)56)39(48-35)46-30-6-4-28(5-7-30)29-14-17-51(18-15-29)25-27-12-19-52(20-13-27)31-8-9-33(44-23-31)40(57)47-34-10-11-36(55)49-41(34)58/h4-9,23-24,27,29,32,34H,2-3,10-22,25-26H2,1H3,(H2,43,56)(H,46,48)(H,47,57)(H,49,55,58)/t32-,34+/m1/s1
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| Chemical Name |
3-[4-[1-[[1-[6-[[(3S)-2,6-dioxopiperidin-3-yl]carbamoyl]pyridin-3-yl]piperidin-4-yl]methyl]piperidin-4-yl]anilino]-5-[(3R)-3-(3-methyl-2-oxoimidazolidin-1-yl)piperidin-1-yl]pyrazine-2-carboxamide
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| Synonyms |
NX-5948; Bexobrutideg; NX 5948; 2649400-34-8; BTK-IN-24; HVD6HGW6JD; NX5948;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 50 mg/mL (61.96 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 3.33 mg/mL (4.13 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 33.3 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. Solubility in Formulation 2: ≥ 3.33 mg/mL (4.13 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 33.3 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 3.33 mg/mL (4.13 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.2392 mL | 6.1961 mL | 12.3922 mL | |
| 5 mM | 0.2478 mL | 1.2392 mL | 2.4784 mL | |
| 10 mM | 0.1239 mL | 0.6196 mL | 1.2392 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Link: https://clinicaltrials.gov/ct2/show/NCT05131022
Conditions:Chronic Lymphocytic Leukemia (CLL)|Small Lymphocytic Lymphoma (SLL)|Diffuse Large B Cell Lymphoma (DLBCL)|Follicular Lymphoma (FL)|Mantle Cell Lymphoma (MCL)|Marginal Zone Lymphoma (MZL)|Waldenstrom Macroglobulinemia (WM)|Primary Central Nervous System Lymphoma (PCNSL)|Secondary Central Nervous System Lymphoma (SCNSL)Link: https://clinicaltrials.gov/ct2/show/NCT07221500
Conditions:Chronic Lymphocytic Leukemia (CLL)|Small Lymphocytic Lymphoma (SLL)Link: https://clinicaltrials.gov/ct2/show/NCT07520006
Conditions:B-cell Lymphoma|Chronic Lymphocytic Leukemia|Small Lymphocytic Lymphoma
Title:Study of NX-5948 Versus Pirtobrutinib in R/R CLL/SLL
Status:Not yet recruiting
updateDate:2026-04-08
Ctid:NCT07516093
Link: https://clinicaltrials.gov/ct2/show/NCT07516093
Conditions:B-cell Lymphoma|Chronic Lymphocytic Leukemia|Small Lymphocytic LymphomaLink: https://clinicaltrials.gov/ct2/show/NCT06717269
Conditions:Healthy VolunteerLink: https://clinicaltrials.gov/ct2/show/NCT06691828
Conditions:Healthy VolunteerProducts are for research use only; We do not sell to patients
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