NSD2-PWWP1 (Kd = 91 ± 8 nM by SPR; IC50 = 104 ± 13 nM by AlphaScreen disrupting H3K36me2 nucleosome interaction; Cellular engagement EC50 = 1.23 ± 0.25 μM by NanoBRET PPI assay). [1]

- UNC6934 binds to the NSD2-PWWP1 domain with a Kd of 91 ± 8 nM, as determined by surface plasmon resonance (SPR). [1]
- UNC6934 disrupts the interaction between NSD2-PWWP1 and nucleosomal H3K36me2 in a dose-dependent manner with an IC50 of 104 ± 13 nM. [1]
- In the presence of salmon sperm DNA (SSD) to block non-specific DNA interactions, UNC6934 effectively disengages full-length NSD2 from nucleosomal H3K36me2 with an IC50 of 78 ± 29 nM, which is ~65-fold more potent than the negative control UNC7145 (IC50 = 5.1 ± 1 μM). UNC6934 was unable to displace full-length NSD2 in the absence of SSD. [1]
- UNC6934 shows no significant effect on NSD2-PWWP1 DNA binding in a fluorescence polarization assay up to 25 μM. [1]
- In cell fractionation experiments, UNC6934 treatment resulted in a modest release of full-length NSD2 from chromatin. [1]
- Following treatment with UNC6934, no changes were observed in global H3K36me2, H3K36me1, H3K36me3, or H3K27me3 levels. [1]
- UNC6934 did not affect loci-specific H3K36me2 levels or mRNA levels of NSD2 target genes, such as adhesion genes. [1]
- UNC6934 did not inhibit any of a panel of 33 methyltransferase domains, including the H3K36 methyltransferases NSD1, NSD2, NSD3, and SETD2. [1]
- No acute cytotoxic effects were observed following treatment of adherent (HCT116, HEK293, HT1080, MCF7, U2OS) or hematopoietic malignancy (KMS-11, MM1S, RS4;11, TKO, UTMC2) cell lines with UNC6934 up to 5 μM over 3-12 days. [1]
- UNC6934 did not affect the proliferation of KMS-11 t(4;14) multiple myeloma cells grown on bone marrow stroma. [1]

- Surface Plasmon Resonance (SPR): Biotinylated NSD2-PWWP1 domain was immobilized on an SA sensor chip. UNC6934 was tested at 2 μM as the highest concentration, with a dilution factor of 0.33 to yield five concentrations. Experiments were performed in single-cycle kinetics with 60 s contact time and 120 s dissociation time at a flow rate of 75 μl min⁻¹ in HBS-EP buffer with 0.5% DMSO. [1]
- Differential Scanning Fluorimetry (DSF): The assay determined the effect of 100 μM UNC6934 on the thermal stability of NSD2-PWWP1 and 15 other PWWP domains. Proteins were used at 0.1 mg ml⁻¹ in buffer (100 mM HEPES, pH 7.5, 150 mM NaCl) containing 5x Sypro Orange. A >2°C increase in Tm was considered binding. UNC6934 did not stabilize the NSD2-PWWP1 F266A mutant. [1]
- dCyper AlphaScreen Binding Assays: A two-phase assay was performed. Phase A: His-tagged NSD2-PWWP1 was titrated against positive (H3K36me2) and negative (H3K36me0) nucleosomes at various NaCl concentrations. Phase B: An optimal NSD2-PWWP1 concentration was used to probe a biotinylated nucleosome panel. For antagonist testing, 5 μl of compound and 5 μl of NSD2-PWWP1 (40 nM) or full-length NSD2 (10 nM) were combined and incubated for 15 min. Then, 5 μl of H3K36me2 dNuc (10 nM) was added and incubated for 30 min. A mixture of nickel-chelate acceptor beads and streptavidin donor beads was added, incubated for 60 min, and the AlphaScreen signal was measured. [1]
- NanoBRET PPI Assay: U2OS cells transfected with H3.3-HaloTag and NSD2-PWWP1-NanoLuc (WT or F266A) or NSD3-PWWP1-NanoLuc were plated in 96-well plates in the presence or absence of compounds for 20 h. After adding the NanoBRET NanoGlo Substrate, donor emission at 450 nm and acceptor emission at 618 nm were measured. A dose-dependent decrease in BRET signal was observed following treatment with UNC6934 (EC50 = 1.23 ± 0.25 μM). [1]
- Fluorescence Polarization DNA Binding/Displacement Assay: A fluorescein-labeled dsDNA (5 nM) and NSD2-PWWP1 (500 nM) were incubated for 30 min with increasing concentrations of UNC6934 (up to 25 μM). The fluorescence polarization signal was measured. UNC6934 showed no significant effect on dsDNA binding to NSD2-PWWP1. [1]
- Crystallography: The structure of NSD2-PWWP1 in complex with UNC6934 was solved (PDB: 6XCG). Crystals were obtained via sitting-drop vapor-diffusion at 18°C in a condition containing 1.6 M (NH₄)₂SO₄, 0.01 M MgCl₂, and 0.1 M HEPES (pH 7.5). [1]

- Cell Culture: HCT116, HEK293, HT1080, MCF7, and U2OS cells were cultured in DMEM with 10% FBS and penicillin/streptomycin. OP9 bone marrow stromal cells were cultured in α-MEM with GlutaMAX, 20% FBS, and 55 μmol liter⁻¹ β-mercaptoethanol. KMS-11 and TKO2 cells were cultured in RPMI with 10% FBS and penicillin/streptomycin. [1]
- Chemical Proteomics: KMS-11 whole-cell lysates (3 mg) were preincubated with DMSO, 20 μM UNC7145, or 20 μM UNC6934 for 1 h. UNC7096-bound magnetic beads were added and incubated for 1 h. Beads were washed, and on-bead digestion was performed with trypsin overnight. The resulting peptides were analyzed by LC-MS/MS. Label-free quantitative MS data were searched using MaxQuant and analyzed with the DEP package in R. [1]
- Fluorescence Microscopy (Immunofluorescence): Cells were fixed with 2% formaldehyde, permeabilized with 0.25% Triton X-100, and blocked with 1% BSA. Cells were incubated overnight with primary antibodies for NSD2 (1:500) and fibrillarin (1:1,000), followed by fluorescent secondary antibodies (1:1,000). DNA was stained with Hoechst 33342. Images were acquired with a confocal microscope. Colocalization was quantified using a CellProfiler pipeline to calculate the Pearson correlation coefficient (PCC). [1]
- Fluorescence Microscopy (Fusion Proteins): U2OS cells were transfected with 0.125 μg of NSD2-GFP and 0.125 μg of fibrillarin-RFP. After 24 h, cells were treated with compound for 4 h or fixed directly. Cells were fixed with 2% formaldehyde and stained with Hoechst 33342. Images were acquired on an EVOS FL Auto 2 Imaging System. [1]
- Western Blotting for Global H3K36me2: Cells were treated with compound for 72 h, collected, and lysed. Samples were boiled in SDS loading buffer and run on NuPAGE gels. Immunoblots were probed with antibodies for H3K36me2 (1:2,000), histone H3 (1:5,000), α-tubulin (1:5,000), and NSD2 (1:1,000), followed by near-infrared detection. [1]
- Cell Viability/Proliferation Assessment (Adherent Cells): Cells (HCT116, HEK293, HT1080, MCF7, U2OS) were treated with 3, 5, or 10 μM of UNC6934 or UNC7145 for 72 h. Cell nuclei were stained and imaged on an IncuCyte live-cell analysis system. [1]
- Cell Viability/Proliferation Assessment (Suspension Cells): Cells (KMS-11, MM1S, RS4;11, TKO, UTMC2) were treated with 3, 5, or 10 μM of UNC6934 or UNC7145 for 6-12 days. Viability was measured using the CellTiter-Glo luminescent cell viability assay. [1]
- KMS-11/OP9 Coculture Proliferation Assay: GFP-expressing KMS-11 and TKO cells were pretreated with 5 μM UNC6934 or UNC7145 for 10 days, then seeded on a confluent monolayer of OP9 bone marrow stromal cells. Proliferation was monitored by counting GFP+ cells over 7 days using an IncuCyte live-cell imaging platform. [1]
- 5-Ethynyl Uridine (5-EU) Incorporation Assay: Cells were pretreated with 5 μM UNC6934 or UNC7145 for 30 min, then fed 5-EU for 45 min. Cells were fixed and stained for nucleolin. Nascent RNA was labeled using a Click-iT kit. Images were acquired and analyzed using a CellProfiler pipeline. [1]
- Reverse Transcription-quantitative PCR (RT-qPCR): Total RNA was isolated and reverse transcribed. qPCR was performed using SYBR Green master mix. Relative transcript levels were determined by the Cₜ method and normalized to B2M or TBP housekeeping genes. [1]
- Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR): Cells (1 x 10⁷) were fixed with 1% paraformaldehyde. Lysed nuclei were treated with micrococcal nuclease and sonicated. Anti-H3K36me2 antibody was added to the chromatin and incubated overnight. DNA was purified and analyzed by qPCR. The percentage of input was calculated. [1]
- Cell Fractionation: KMS-11 cell pellets were resuspended in Hypotonic Buffer A. The supernatant (cytoplasmic fraction) was collected. The pellet was resuspended in Buffer B without NaCl, followed by Buffer B with 150 mM NaCl to extract the nucleoplasmic fraction. The remaining chromatin-bound pellet was resuspended in histone buffer. Fractions were analyzed by western blot for NSD2. [1]

- UNC6934 showed no acute cytotoxic effects in adherent (HCT116, HEK293, HT1080, MCF7, U2OS) or hematopoietic malignancy (KMS-11, MM1S, RS4;11, TKO, UTMC2) cell lines up to 5 μM over 3-12 days. [1]
- UNC6934 (10 μM) showed minimal off-target activity in a panel of 90 central nervous system receptors, channels, and transporters, inhibiting only two targets >50%. The human sodium-dependent serotonin transporter was the only protein inhibited with a measurable Ki of 1.4 ± 0.8 μM. [1]

[1]. A chemical probe targeting the PWWP domain alters NSD2 nucleolar localization. Nat Chem Biol. 2022 Jan;18(1):56-63.

- Background: NSD2 is the primary enzyme responsible for H3K36me2, a mark associated with active gene transcription. It is aberrantly expressed in multiple cancers, including multiple myeloma (MM) via t(4;14) translocation. UNC6934 is a chemical probe for the NSD2-PWWP1 domain. [1]
- Mechanism of Action: UNC6934 occupies the canonical methyl-lysine binding pocket (aromatic cage composed of Y233, W236, and F266) of the NSD2-PWWP1 domain, competing directly with H3K36me2. This binding induces a conformational rearrangement of three side chains (Y233, F266, E272). The cyclopropyl ring of UNC6934 fits tightly in the aromatic cage, and a bulkier isopropyl group (as in UNC7145) results in no appreciable binding. [1]
- Selectivity: UNC6934 is selective for NSD2-PWWP1 over 15 other human PWWP domains. The selectivity is explained by structural differences, such as G268 in NSD2 at the bottom of a cavity accommodating the benzoxazine ring. Replacing this with serine in NSD3 would occlude ligand binding. [1]
- Cellular Target Engagement: A biotin-labeled affinity reagent (UNC7096) based on UNC6934 enriched endogenous NSD2 from cell lysates, an effect blocked by UNC6934 but not UNC7145. Proteomic analysis identified NSD2 as the only protein significantly depleted by competition with UNC6934. [1]
- Phenotypic Effect: UNC6934 promotes accumulation of endogenous NSD2 in the nucleolus, phenocopying the effect of PWWP1-truncating mutations in t(4;14) MM. This is due to a competitive mechanism between NSD2's chromatin reader domains and C-terminal nucleolar localization sequences (NoLSs). [1]

C24H21N5O4

443.454644918442

443.16

C, 65.00; H, 4.77; N, 15.79; O, 14.43

2561494-77-5

146676966

Off-white to gray solid powder

1.8

2

6

6

33

736

0

O=C(C1C=CC2=C(C=1)OCC(N2)=O)N(CC1C=CC(C(NC2C=CN=CN=2)=O)=CC=1)C1CC1

KOZGEDUWAQFVAV-UHFFFAOYSA-N

InChI=1S/C24H21N5O4/c30-22-13-33-20-11-17(5-8-19(20)27-22)24(32)29(18-6-7-18)12-15-1-3-16(4-2-15)23(31)28-21-9-10-25-14-26-21/h1-5,8-11,14,18H,6-7,12-13H2,(H,27,30)(H,25,26,28,31)

N-Cyclopropyl-3-oxo-N-(4-(pyrimidin-4-ylcarbamoyl)benzyl)-3,4-dihydro-2H-benzo[b][1,4]oxazine-7-carboxamide

UNC6934; UNC-6934; UNC6,934; 2561494-77-5; N-cyclopropyl-3-oxo-N-(4-(pyrimidin-4-ylcarbamoyl)benzyl)-3,4-dihydro-2H-benzo[b][1,4]oxazine-7-carboxamide; CHEMBL5200945; N-cyclopropyl-3-oxo-N-({4-[(pyrimidin-4-yl)carbamoyl]phenyl}methyl)-3,4-dihydro-2H-1,4-benzoxazine-7-carboxamide; UNC 6934;

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 : 25 mg/mL (56.38 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.64 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.64 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 25.0 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.64 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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