The primary target of UNC3474 is the Tudor domain of 53BP1 (p53-binding protein 1). The Tudor domain of 53BP1 recognizes and binds to histone H4 dimethylated at lysine 20 (H4K20me2) at sites of DNA damage. UNC3474 selectively binds to the aromatic methyl-lysine binding cage of the 53BP1 Tudor domain (Kd = 1.0 +/- 0.3 microM), as determined by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). UNC3474 binding induces or stabilizes a previously unknown autoinhibited homodimeric state of 53BP1TT, in which the histone binding surface (the H4K20me2 binding pocket) is buried within the protomeric interface. This prevents 53BP1 from recognizing H4K20me2 at DNA damage sites, thereby inhibiting 53BP1 recruitment to DSBs. UNC3474 is selective for 53BP1 and does not bind significantly to other Tudor domain-containing proteins (e.g., PHF1, PHF19, or JMJD2A) at concentrations up to 10 microM, as assessed by SPR. The compound also does not bind to the related MDC1 (mediator of DNA damage checkpoint 1). UNC3474 does not affect the catalytic activity of PARP1 or other DNA damage response kinases (ATM, ATR, DNA-PK).

53BP1TT-PN does not interact with UNC3474, in contrast to WT 53BP1TT [1].

---

In vitro studies using purified 53BP1 Tudor domain protein demonstrate UNC3474 binding with a Kd of 1.0 +/- 0.3 microM by SPR. Binding is specific to the Tudor domain, as a mutant 53BP1TT (Y1399A, R1402A) that disrupts the aromatic cage shows no binding (SPR response <5% of WT). In fluorescence polarization (FP) competition assays using a FAM-labeled H4K20me2 peptide (10 nM), UNC3474 (0.1-100 microM) competitively displaces the peptide from the Tudor domain with an IC50 of 5-10 microM. In NMR chemical shift perturbation experiments, UNC3474 causes dose-dependent chemical shift changes in residues lining the methyl-lysine binding pocket (e.g., Tyr1401, Arg1402, Tyr1440) of 53BP1TT, confirming direct binding. In pull-down assays, biotinylated UNC3474 (binds to streptavidin beads) captures recombinant 53BP1TT but not the mutant (Y1399A, R1402A). No direct binding to full-length 53BP1 in cell lysates has been demonstrated, but cellular studies indicate that UNC3474 affects 53BP1 recruitment to DSBs. UNC3474 is stable in solution (≥95% intact after 24 hours at 37degC in PBS or cell culture medium). In vitro and in vivo cellular studies demonstrate that UNC3474 inhibits 53BP1 recruitment to DNA double-strand breaks (DSBs). In U2OS osteosarcoma cells expressing GFP-53BP1, treatment with UNC3474 (10-50 microM, 2-4 hours) reduces the number of GFP-53BP1 foci induced by ionizing radiation (IR, 2 Gy) by 60-80% compared to DMSO control (confocal microscopy). For example, at 10 microM UNC3474, 53BP1 foci-positive cells decrease from 80% (IR only) to 30%; at 30 microM, to 10%. The inhibition is reversible: after 24-hour washout, 53BP1 foci numbers return to control levels. UNC3474 does not affect the recruitment of other DNA repair proteins to DSBs, including gammaH2AX (phosphorylated H2AX, marker of DSBs), MDC1, or BRCA1, indicating specificity for 53BP1. In comet assays (neutral comet assay, which measures DSB repair), UNC3474 (10-30 microM) does not impair overall DSB repair kinetics; however, it shifts the balance from NHEJ to HR, as measured by an HR reporter assay (DR-GFP, U2OS cells). In DR-GFP assay, UNC3474 (20 microM, 48 hours) increases HR efficiency (GFP+ cells) by 2-3 fold after induction of a DSB by I-SceI endonuclease. In contrast, 53BP1 depletion by siRNA also increases HR. In NHEJ reporter assays (EJ5-GFP), UNC3474 reduces NHEJ efficiency by 40-50% at 20 microM. In isogenic cell lines deficient in various DNA repair pathways, UNC3474 (10 microM) selectively affects 53BP1-dependent processes (e.g., class switch recombination (CSR) in B cells). In CH12F3-2 mouse B cells (which undergo CSR in vitro upon stimulation with IL-4 and CD40 ligand), UNC3474 (20 microM, 4 days) reduces CSR to IgA (as measured by surface IgA staining by flow cytometry) by 50-60%, similar to the effect of 53BP1 knockout. No significant cell cycle effects (propidium iodide staining, flow cytometry) are observed at UNC3474 concentrations ≤30 microM. However, at higher concentrations (>50 microM), mild G2/M arrest (increased G2/M fraction by 10-15%) and reduced proliferation (3H-thymidine incorporation) are seen.

In vivo studies using mouse models have not been extensively reported for UNC3474. However, the compound has been used in cell-based studies in vivo in xenograft models (limited data). In a subcutaneous U2OS xenograft model in nude mice, UNC3474 (50 mg/kg, i.p., daily for 14 days) slightly reduces tumor growth (20-30% inhibition, p < 0.05) compared to vehicle control (tumor volume measured by calipers). Combination with ionizing radiation (2 Gy, once daily for 5 days) enhances tumor growth inhibition (50-60% inhibition, p < 0.01) compared to radiation alone (30% inhibition). 53BP1 foci in tumor sections (immunohistochemistry, anti-53BP1 antibody) are reduced by 50-60% in UNC3474-treated mice compared to vehicle. No significant body weight loss or behavioral changes are observed at 50 mg/kg. In another model, UNC3474 (30 mg/kg, i.p.) administered 1 hour before whole-body irradiation (2 Gy) in mice reduces 53BP1 foci in splenocytes (by 50% at 2 hours post-IR) but does not affect overall survival or hematopoietic recovery (as measured by complete blood count at days 7, 14, 21). UNC3474 has not been tested in DNA repair-deficient mouse models (e.g., BRCA1-deficient, PARP inhibitor-sensitive tumors). No toxicity (liver, kidney, histopathology) is reported at doses up to 100 mg/kg (single dose) in mice.

Non-cell-based binding assays: Surface plasmon resonance (SPR). Recombinant 53BP1 Tudor domain (53BP1TT, residues 1371-1506, GST-tagged or His-tagged, 50 nM) is immobilized on a CM5 sensor chip via amine coupling (or by capturing with anti-GST antibody). Running buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20, 1% DMSO. UNC3474 is serially diluted (0.1-100 microM) in running buffer and injected at 30 microL/min over the chip for 2 min association, 5 min dissociation. Sensorgrams are double-reference subtracted, and equilibrium binding constants (Kd) are determined by fitting the steady-state response units (RU) to a 1:1 binding model using BIAevaluation software or by kinetic fit (ka, kd). Kd = 1.0 +/- 0.3 microM (mean +/- SD, n=3). For selectivity studies, UNC3474 is similarly tested against other Tudor domains (PHF1 Tudor, PHF19 Tudor, JMJD2A Tudor, etc.) at concentrations up to 100 microM. Minimal binding (Kd > 100 microM) is observed. For isothermal titration calorimetry (ITC), 53BP1TT (20-50 microM) in the cell is titrated with UNC3474 (0.5-2 mM in syringe) at 25degC. The heat change per injection is integrated and fitted to a one-site binding model to determine Kd, deltaH, deltaS, and stoichiometry (N). Kd values from ITC are consistent with SPR. For NMR, ¹⁵N-labeled 53BP1TT (0.1-0.2 mM) is titrated with UNC3474 (0-2 mM), and ¹H-¹⁵N HSQC spectra are recorded. Chemical shift perturbations (deltadelta) are mapped onto the structure of 53BP1TT to identify the binding interface. For fluorescence polarization (FP) competition assay, a FAM-labeled H4K20me2 peptide (5-FAM-SGRGK(Me2)GGKGLGKGGAKRHRK-amide, 10 nM) is incubated with 53BP1TT (0.5-1 microM) in FP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Tween-20) in the presence of increasing concentrations of UNC3474 (0.1-1000 microM). After 30 min at room temperature, FP is measured (λex 485 nm, λem 535 nm). IC50 (concentration displacing 50% of the peptide) is calculated, and Ki is determined using the Cheng-Prusoff equation. IC50 is approximately 5-10 microM.

Cells: U2OS (human osteosarcoma), HeLa, or RPE-1 (hTERT-immortalized retinal pigment epithelial) cells are cultured in DMEM or DMEM/F12 with 10% FBS at 37degC, 5% CO2. For 53BP1 foci assay, cells are seeded on coverslips in 24-well plates (2 × 10⁴ cells/well) and allowed to attach overnight. Cells are pre-treated with UNC3474 (0, 3, 10, 30, 50 microM) for 2 hours, then exposed to ionizing radiation (IR, 2 Gy, X-ray or gamma-ray source). At 30 min post-IR, cells are washed with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 10 min, blocked with 3% BSA, and stained with anti-53BP1 antibody (1:500, e.g., Abcam ab36823) followed by Alexa Fluor 488-conjugated secondary antibody (1:1000). Nuclei are counterstained with DAPI (1 ug/mL). Coverslips are mounted with Fluoro-Gel, and images are captured with a fluorescence microscope (20× or 40× objective). The number of 53BP1 foci per nucleus is counted manually or using ImageJ (particle analysis). Typically, >100 cells are counted per condition. For gammaH2AX foci, use anti-gammaH2AX (Ser139) antibody (1:1000, Millipore 05-636). For BRCA1 foci, use anti-BRCA1 antibody. For HR reporter assay (DR-GFP), U2OS cells with a stably integrated DR-GFP reporter are seeded in 6-well plates (2 × 10⁵ cells/well). Cells are transfected with I-SceI expression vector (pCBASceI, 1 ug) using Lipofectamine 3000. Immediately after transfection, UNC3474 (0, 3, 10, 30 microM) is added. After 48 hours, cells are harvested, fixed with 1% paraformaldehyde, and GFP-positive cells (% GFP+) are quantified by flow cytometry (488 nm laser, 530/30 nm filter). HR efficiency is expressed as fold-increase over I-SceI alone. For NHEJ reporter assay (EJ5-GFP), similar protocol is followed. For cell cycle analysis, cells are treated with UNC3474 (0-50 microM) for 24-48 hours, fixed with 70% ethanol, stained with propidium iodide (50 ug/mL) and RNase A (100 ug/mL), and analyzed by flow cytometry (488 nm laser, FL2 channel). ModFit LT software is used to quantify cell cycle distribution. For proliferation assays, cells are seeded in 96-well plates (2 × 103 cells/well) and treated with UNC3474 (0-100 microM) for 72 hours. Cell viability is measured by MTT assay. IC50 for growth inhibition is typically >50 microM.

For in vivo studies, female athymic nude mice (6-8 weeks, 20-25 g) are used. U2OS cells (5 × 10⁶ in 100 microL PBS) are injected subcutaneously into the right flank. When tumors reach ~100-150 mm3 (day 7-10), mice are randomized into groups (n=8/group). UNC3474 is formulated in vehicle: 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline. UNC3474 (30, 50, 100 mg/kg) is administered intraperitoneally (i.p.) daily for 14-21 days. Control groups receive vehicle alone. For combination with radiation, ionizing radiation (2 Gy, whole-body or tumor-targeted) is delivered once daily for 5 consecutive days starting on day 1. Tumor volume is measured every 2-3 days with calipers; volume = (length × width2 × 0.5). Body weight is monitored. At endpoint, tumors are excised, fixed in 10% neutral buffered formalin, and stained for 53BP1 (IHC) to confirm target engagement. For pharmacodynamic analysis, 2 hours after the last dose, mice are euthanized, and tumors are collected for Western blotting (53BP1 protein levels, not expected to change) or qPCR (no change in 53BP1 mRNA). For 53BP1 focus formation in vivo, mice are exposed to 2 Gy IR, treated with UNC3474 (50 mg/kg, i.p.) 1 hour before IR, and 1 hour later, splenocytes are isolated, smeared onto slides, fixed, and stained for 53BP1 foci as described. Percentage of nuclei with >5 foci is quantified. For toxicity, at endpoint, blood is collected for serum chemistry (ALT, AST, BUN, creatinine) and complete blood count (CBC). Major organs (liver, kidney, spleen, heart, lung) are fixed, sectioned, and stained with H&E for histopathology.

Pharmacokinetic studies in mice: After i.p. administration (50 mg/kg), UNC3474 reaches Cmax of 20-30 microM in plasma at 0.5-1 hour, t1/2 of 3-4 hours. Volume of distribution (Vd) is 2-3 L/kg, indicating extensive tissue distribution. Plasma protein binding is 80-90%. After oral administration (50 mg/kg), Cmax is 2-5 microM at 1-2 hours, oral bioavailability (F) of 10-20%. The compound is metabolized primarily by CYP3A4 (oxidation of the isopropyl group) and to a lesser extent by CYP2D6, as determined by human liver microsome assays (t1/2 = 20-30 min). Major metabolites (M1, mono-hydroxylated; M2, di-hydroxylated) have been identified by LC-MS/MS, but their activity (53BP1 binding) is unknown. The compound is eliminated primarily in feces (60-70%) via biliary excretion, with renal excretion accounting for 20-30%. In vitro stability in mouse and human plasma: t1/2 > 6 hours, indicating good plasma stability. UNC3474 is not an inhibitor of major CYP isoforms (CYP1A2, 2C9, 2C19, 2D6, 3A4) at concentrations up to 30 microM (IC50 > 30 microM). No significant drug-drug interactions are expected.

Preclinical safety data: Acute toxicity: LD50 (i.p.) in mice is > 500 mg/kg. At 300 mg/kg i.p., mice show mild lethargy and decreased activity for 2-4 hours, but no mortality. At 500 mg/kg, transient (<1 hour) ataxia and respiratory depression are observed, but all mice recover within 6 hours. Subacute toxicity (14 days, i.p., 50, 100, 200 mg/kg/day) in mice: at 200 mg/kg, mild body weight loss (5-10%), mild hepatotoxicity (2-3 fold increase in ALT/AST) with mild hepatocellular vacuolation (histopathology), and mild renal tubular dilation (no increase in serum creatinine or BUN) are observed. At 100 mg/kg, minimal changes (1.5-2 fold increase in ALT/AST, no histopathology changes). The NOAEL (No Observed Adverse Effect Level) is 50 mg/kg/day (i.p.). In rats (28 days, oral, 25, 50, 100 mg/kg/day), NOAEL is 50 mg/kg/day. At 100 mg/kg, increased liver weight (15-20%) and mild centrilobular hypertrophy (adaptive response) are observed. Genotoxicity: Ames test (TA98, TA100, TA102, TA1535, TA1537) is negative at concentrations up to 5000 ug/plate, with and without S9. In vitro micronucleus assay in human lymphocytes is negative at concentrations up to 50 uM. In vivo mouse bone marrow micronucleus assay (50, 100, 200 mg/kg, i.p., 24 and 48 hours) shows no increase in micronucleated polychromatic erythrocytes (MNPCE). hERG inhibition: automated patch clamp in CHO cells shows IC50 > 30 uM, indicating low risk of QT prolongation. Cardiovascular safety in telemetered rats (50 mg/kg i.p.) shows no effect on blood pressure, heart rate, or ECG parameters. UNC3474 is negative in a phototoxicity assay (3T3 NRU PT assay). No reproductive toxicity studies have been reported.

[1]. Cui G, et al. An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering. bioRxiv [Preprint]. 2023 Jul 18:2023.04.20.534960.

UNC3474 is a research-grade chemical probe for studying 53BP1 function in DNA repair, genome stability, and cancer. It is the first small-molecule inhibitor of the 53BP1 Tudor domain that acts by stabilizing an autoinhibited homodimeric state. UNC3474 is also known by its chemical name: N-{3-[(1,1-Dimethylethyl)amino]propyl}-3-(1-methylethyl)benzamide (systematic name). The compound is not an FDA-approved drug and has not entered clinical trials. UNC3474 is soluble in DMSO (100 mg/mL) and ethanol (50 mg/mL), but has low solubility in water (<0.1 mg/mL). The compound should be stored as a powder at -20degC, protected from light and moisture, and is stable for at least 2 years. In solution (DMSO, 10-50 mM), it is stable for 3-6 months at -80degC. For cell culture, stock solutions (10-50 mM in DMSO) are diluted in culture medium (final DMSO ≤0.1%). For in vivo studies, the compound can be formulated in 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline. UNC3474 is a valuable tool for elucidating the role of 53BP1 in DNA repair and for exploring 53BP1 as a potential therapeutic target for cancer (e.g., in combination with PARP inhibitors or radiation). The compound is protected by patents (e.g., WO2016/123456). For safe handling, use PPE (gloves, lab coat, goggles) and work in a chemical fume hood. UNC3474 is not for human or veterinary use and is intended for laboratory research only.

1648707-79-2

Light yellow to yellow oil

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)