Nuclear binding SET domain 1 (NSD1) – an H3K36 methyltransferase [1]. NSD-IN-2 targets Nuclear receptor-binding SET Domain protein 1 (NSD1), a histone H3 lysine 36 (H3K36) methyltransferase. It acts as a potent and irreversible inhibitor of NSD1
Nuclear receptor-binding SET Domain protein 1 (NSD1) – a histone H3 lysine 36 (H3K36) methyltransferase. It acts as a covalent, irreversible inhibitor targeting cysteine C2062 in the autoinhibitor loop of the NSD1 SET domain. Also shows weaker, slower covalent engagement with NSD3 (kinact/KI = 3.8 M⁻¹s⁻¹) and minimal engagement with NSD2 [2].

In primary mouse knee chondrocytes, treatment with NSD-IN-2 (0.9 μM) significantly reduced the enrichment of NSD1 and H3K36me2 on the KLF9 promoter, as measured by chromatin immunoprecipitation (ChIP) assay. This inhibition subsequently decreased KLF9 and ATG14 expression at both the mRNA and protein levels [1].

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In Vitro: BT5 covalently engages with the NSD1 SET domain, with 80% engagement after 8 h incubation at 32°C. The second-order rate constant for covalent binding is kinact/KI = 19.6 M⁻¹s⁻¹ (KI = 25.0 μM, kinact = 4.8 × 10⁻⁴ s⁻¹). In histone methyltransferase (HMT) assays using nucleosomes as substrate, BT5 inhibits NSD1 activity with IC50 = 5.8 μM after 4 h incubation and IC50 = 1.4 μM after 16 h incubation [2].
BT5 also inhibits NSD2 and NSD3 at higher concentrations (4 h incubation), likely via non-covalent binding. In a panel of other histone methyltransferases (including ASH1L, DOT1L, EZH2, G9a, GLP, PRDM9, PRMT1, PRMT3, PRMT4, SETD2, SET7/9, SMYD1, SMYD2, SUV39H2), no significant inhibition was detected at 50 μM BT5. No binding to ASH1L SET domain was observed. In broader selectivity panels, BT5 (50 μM) showed no significant activity against 10 HDACs, 4 sirtuins, 6 HATs, or a panel of 291 protein kinases [2].

Enzyme Assay: Radiometric histone methyltransferase (HMT) assays were performed. Recombinant NSD1 SET domain (200 nM) was incubated with BT5 at indicated concentrations for specified times (4 h or 16 h) at 32°C. The reaction was initiated by adding 250 nM chicken nucleosomes, 0.4 μM ³H-labeled S-adenosyl methionine (SAM), and 2.4 μM unlabeled SAM. After 1 h at room temperature, the reaction was quenched with 10% trichloroacetic acid (TCA). Samples were transferred to 96-well filter plates, washed with TCA and ethanol, dried, and mixed with scintillant. Radiometric signal was read on a microplate counter. IC50 values were calculated using GraphPad Prism [2].
For covalent binding kinetics, MS experiments were performed. NSD1 (1 μM) was incubated with various concentrations of BT5 (4–48 μM) in PBS buffer with 1 mM DTT and 2 μM SAM at 30°C for time periods ranging from 0.5 to 8 h. Reactions were quenched with 0.2% formic acid. LC-MS analysis was carried out on a Q-TOF LC/MS. Relative peak heights at the unmodified protein weight and protein-compound adduct weight were compared to obtain the percent covalent engagement. The kinact/KI was determined by measuring the rate of formation of the ligand-NSD1 complex as a function of ligand concentration [2].
X-ray crystallography was used to determine the structure of the NSD1 SET domain in complex with covalently bound BT3 (a thiocyanate analog). NSD1 SET (residues 1863-2085) at 15.5 mg/mL was incubated with 5-fold molar excess of BT3 for 2 h on ice. Crystals were grown by sitting drop vapor diffusion at 17°C using a reservoir solution of 100 mM sodium cacodylate (pH 6.5), 200 mM calcium acetate, and 18% PEG 8000. Data were collected at the Advanced Photon Source and structures were determined by molecular replacement and refined using PHENIX [2].

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No classical enzyme assay for NSD-IN-2 was performed. Instead, its inhibitory effect on NSD1 was assessed via a ChIP assay. Cells were treated with NSD-IN-2 (0.9 μM) or an equal volume of DMSO (control). After treatment, protein-DNA crosslinking was performed using 1% formaldehyde. Chromatin was fragmented, and immunoprecipitation was carried out using antibodies against NSD1 and H3K36me2, with normal IgG as a negative control. The enrichment of NSD1 and H3K36me2 on the KLF9 promoter was then quantified by qRT-PCR [1].

Cell Assay: For cellular thermal shift assay (CETSA), HEK293T cells were transfected with Flag-NSD1 SET construct. Cells were treated with DMSO or 5 μM BT5 overnight (~16 h). Cells were harvested, resuspended in PBS, and aliquots were heated at different temperatures for 3 min followed by cooling. Cells were lysed by freeze-thaw cycles, centrifuged, and supernatants were analyzed by SDS-PAGE and western blot using anti-FLAG antibody. BT5 stabilized NSD1 SET by ~4°C compared to DMSO [2].
For HiBiT CETSA, HEK293T cells were transfected with N-HiBiT-NSD1 construct, treated with DMSO or 5 μM BT5, and subjected to thermal shift analysis using Nano-Glo HiBiT Lytic Detection System. BT5 stabilized NSD1 SET by ~4°C [2].
For immunoblotting, NUP98-NSD1 and HM-2 cells were treated with indicated doses of BT5 for 8 days (with media and compound refresh on day 4). Whole cell lysates were prepared and analyzed by western blot for H3K36me2, H3K36me3, H3K27me3, H3K79me3, total H3, and actin. BT5 caused dose-dependent suppression of H3K36me2 only in NUP98-NSD1 cells, with no effect on other marks [2].
For gene expression analysis, NUP98-NSD1 cells were treated with BT5 for 8 days. RNA was isolated, reverse transcribed to cDNA, and qPCR was performed using Taqman probes for Hoxa9, Hoxa5, Hoxa7, and Meis1. BT5 treatment caused up to 50% reduction in expression of these NUP98-NSD1 target genes [2].
For chromatin immunoprecipitation (ChIP), NUP98-NSD1 cells were treated with DMSO or 2 μM BT5 for 4 days. Cells were fixed with 1% paraformaldehyde, and DNA-protein complexes were immunoprecipitated using antibodies against H3K36me2, H3K36me3, H3, or normal IgG. DNA was eluted and subjected to qPCR using primers for the Hoxa9 promoter region. BT5 reduced H3K36me2 level at the Hoxa9 promoter [2].
For cell viability/growth inhibition, murine bone marrow-derived cells (NUP98-NSD1, NUP98-HOXA9, MOZ-TIF2, HM-2) and human leukemia cells (K562, SET2, MOLM13) were cultured and treated with varying concentrations of BT5 for 7 days (with media and compound refresh on day 4). MTT assay was performed to assess viability. GI50 values were calculated. BT5 showed pronounced growth inhibition in NUP98-NSD1 cells (GI50 = 0.87 ± 0.09 μM at day 7), while other cell lines were 6-8 fold less sensitive (GI50 = 5.1–7.7 μM at day 7) [2].
For colony formation assays, primary patient samples (NUP98-NSD1 or MLL-ENL) and normal CD34+ bone marrow progenitor cells were cultured in methylcellulose medium supplemented with cytokines. Cells were treated with BT5 (0–12 μM) for 10 days, and colonies were counted. BT5 reduced colony formation in NUP98-NSD1 sample in a dose-dependent manner but had no effect on MLL-ENL sample or normal CD34+ cells up to 12 μM [2].

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Primary mouse knee chondrocytes were separated and cultured. NSD-IN-2 was supplemented into the chondrocyte culture at a concentration of 0.9 μM, with an equal amount of dimethylsulfoxide (DMSO) used as the control. After treatment, the expression of relevant genes (KLF9 and ATG14) was verified by qRT-PCR and Western blot analysis. Additionally, ChIP assays were performed on these treated cells to detect the enrichment of NSD1 and H3K36me2 on the KLF9 promoter [1].

Stability was assessed by NMR in two different buffers, including the one used for the HMT assay; BT5 remained unaffected for at least 1-2 days under these conditions [2].

BT5 at 12 μM showed no toxicity in normal CD34+ bone marrow progenitor cells in colony formation assays. In HEK293T cells, no overt toxicity was reported at 5 μM for CETSA experiments. The aziridine moiety in BT5 may confer some non-selective reactivity; however, incubation of BT5 with CDC25B phosphatase and thioredoxin (both containing reactive cysteines) showed no or minimal covalent binding. At concentrations exceeding 5 μM, BT5 is likely associated with some toxicity, as evidenced by growth inhibition in non-NUP98-NSD1 cell lines (GI50 = 5.1–7.7 μM) [2].

[1]. Nuclear binding SET domain 1 alleviates cartilage ferroptosis in knee osteoarthritis by upregulating the krüppel-like factor 9/autophagy-related 14 pathway via H3K36me2 modification. J Cell Commun Signal. 2025 Jun 29;19(3):e70027.

[2]. Covalent inhibition of NSD1 histone methyltransferase. Nat Chem Biol. 2020 Dec;16(12):1403-1410

[3]. Nsd family inhibitors and methods of treatment therewith. US20190183865A1.

NSD-IN-2 is used as a pharmacological inhibitor of NSD1 to validate the role of NSD1 in the H3K36me2/KLF9/ATG14 signaling axis in chondrocytes. The study demonstrated that NSD1 enhances KLF9 expression to increase ATG14 expression via H3K36me2 modification, thereby relieving knee osteoarthritis cartilage ferroptosis. NSD-IN-2 treatment was employed to suppress NSD1 activity, which led to reduced KLF9 and ATG14 expression, confirming the regulatory relationship [1].

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BT5 is a first-in-class, irreversible small molecule inhibitor of the NSD1 SET domain, developed through NMR fragment screening and chemical optimization. It contains a methyl-aziridine warhead that covalently targets C2062 in the autoinhibitor loop of NSD1. The crystal structure of NSD1 with covalently bound BT3 (a thiocyanate analog) revealed that binding induces a conformational change in the autoinhibitory loop, forming a channel-like pocket suitable for small molecule targeting [2].
BT5 demonstrates on-target activity in NUP98-NSD1 leukemia cells, including inhibition of H3K36 dimethylation, downregulation of target genes (Hoxa9, Hoxa5, Hoxa7, Meis1), and impaired colony formation in a NUP98-NSD1 patient sample, with no effect on MLL-ENL sample or normal CD34+ cells. This study provides a proof-of-concept that targeting NSD SET domains with small molecules is feasible and facilitates development of next-generation inhibitors [2].

C10H11N3OS

221.28

221.06

C, 54.28; H, 5.01; N, 18.99; O, 7.23; S, 14.49

2351225-46-0

138713923

White to light yellow solid at room temperature

2

2

5

1

15

265

0

CC1CN1C2=CC(=C3C(=C2)SC(=N3)N)O

PHBIAWCGMOCBGF-UHFFFAOYSA-N

InChI=1S/C10H11N3OS/c1-5-4-13(5)6-2-7(14)9-8(3-6)15-10(11)12-9/h2-3,5,14H,4H2,1H3,(H2,11,12)

2-amino-6-(2-methylaziridin-1-yl)-1,3-benzothiazol-4-ol

BT5; 2-Amino-6-(2-methylaziridin-1-yl)-1,3-benzothiazol-4-ol; 2351225-46-0; NSD1 inhibitor BT5; SCHEMBL21091275

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)

DMSO: ~100 mg/mL (451.9 mM)

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.

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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).

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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)

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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).

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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

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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.

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