NSD3-PWWP1 Antagonist
BI-9321
BI-9321 is a potent, highly selective antagonist of the PWWP1 domain of NSD3. This first-in-class chemical probe has shown an in vitro potency of 200 nM and cellular target engagement at around 1 nM. It is suitable for in vitro experiments. Myc mRNA downregulation and reduced proliferation was observed in MOLM-13 cells treated with BI-9321.
More information
Human NSD3 is encoded by the WHSC1L1 gene, located in the 8p11-p12 amplicon, which is frequently amplified in cancer different tumor types. The methyltransferase NSD3 is a multi-domain epigenetic regulator that exists in three isoforms (long, short and the testis-specific Whistle). Both NSD3 long and Whistle isoforms contain the SET domain, with lysine methyltransferase activity, as well as several chromatin binder motifs so called “reader domains”, including PHD and two PWWP domains named PWWP1 and PWWP2. The NSD3-short isoform contains only the first PWWP domain.
Initial binders to the proposed methyl-lysine binding site of the PWWP1 domain of NSD3 were identified applying fragment based screening (FBS) methods. Consecutively, structure-based optimization yielded in BI-9321, a potent antagonist of the PWWP1 (Pro-Trp-Trp-Pro) domain of NSD3 (Nuclear Receptor Binding SET Domain 3). High selectivity of BI-9321 was confirmed using in vitro assays and quantitative chemical proteomics. Cellular target engagement was confirmed with FRAP (Fluorescence Recovery After Photobleaching) and BRET (Bioluminescence Resonance Energy Transfer) at 1 µM.
BI-9321 was discovered in collaboration with the Structural Genomics Consortium (SGC).
BI-9321 in complex with NDS3
| Probe name / negative control | BI-9321 | BI-9466 |
| MW [Da, free base]a | 360.4 | 295.4 |
| TR-FRETb (IC50) [nM] | 203 | 120 000 |
| SPR (Kd)c [nM] | 166 | 144 000 |
| ITC (Kd)d [nM] | 445 | n.d. |
a For the salt form you will get, please refer to the label on the vial and for the molecular weight of the salt, please refer to the FAQs
b 1x PBS; 0.05% Tween20; 0.1 % BSA; filtered
c 50 mM TRIS, pH 8.0; 150 mM NaCl; 1 mM TCEP; 0.005 % Tween 20; 2% DMSO
d 20 mM HEPES, 100 mM NaCl, 3% DMSO, pH 8.0
| Probe name / negative control | BI-9321 | BI-9466 |
| logD @ pH 11 | 1.5 | -0.16 |
| Solubility @ pH 6.8 [µg/mL] | >100 | n.d. |
| Caco-2 permeability AB @ pH7.4 [*10-6 cm/s] | 16 | n.d. |
| Caco-2 efflux ratio | 2.8 | n.d. |
| Microsomal stability (human/mouse/rat) [% QH] | <23 / <24 / <24 | n.d. |
| Hepatocyte stability (human/mouse/rat) [% QH] | <10 / n.d. / 31 | n.d. |
| Plasma Protein Binding (human/mouse/rat) [%] | 41.7 / 43.5 / 45.5 | n.d. |
| CYP 3A4 (IC50) [µM] | 19 | n.d. |
| CYP 2C8 (IC50) [µM] | 5.4 | n.d. |
| CYP 2C9 (IC50) [µM] | 1.3 | n.d. |
| CYP 2C19 (IC50) [µM] | <0.2 | n.d. |
| CYP 2D6 (IC50) [µM] | 23 | n.d. |
BI-9466 is a closely related analogue of BI-9321, exhibiting a more than 500 fold weaker affinity as determined by TR-FRET and SPR. No target engagement up 100 µM could be observed with protein and ligand based BRET assays.
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BI-9466 which serves as a negative control
A kinase panel is available on opnMe.com. BI-9321 did not hit any of the 31 tested kinases.
| SELECTIVITY DATA AVILABLE | BI-9321 | BI-9466 |
SafetyScreen44™ with kind support of  | Yes | Yes |
| Invitrogen® | Yes | No |
| DiscoverX® | No | No |
| Dundee | No | No |
Download selectivity data:
BI-9321_selectivityData_0.xlsx
BI-9466_selectivityData.xlsx
The X-ray crystal structure of NSD3-PWWP1 in complex with BI-9321 is available (PDB code: 6G2O)1.
BI-9321 is a potent first in class chemical probe, antagonizing the PWWP1 domain of NSD3 in cells.
Fragment-based discovery of a chemical probe for the PWWP1 domain of NSD3
Böttcher J., Dilworth D., Reiser U., Neumüller RA., Schleicher M., Petronczki M., Zeeb M., Mischerikow N., Allali-Hassani A., Szewczyk MM., Li F., Kennedy S., Vedadi M. Barsyte-Lovejoy D., Brown PJ., Huber KVM., Rogers CM., Wells CI., Fedorov O., Rumpel K., Zoephel A., Mayer M., Wunberg T., Böse D., Zahn S., Arnhof H., Berger H., Reiser C. Hörmann A., Krammer T., Corcokovic M., Sharps B., Winkler S., Häring D., Cockcroft XL., Fuchs JE., Müllauer B., Weiss-Puxbaum A., Gerstberger T., Boehmelt G., Vakoc CR., Arrowsmith CH., Pearson M., McConnell DB.
Nature Chemical Biology 2019, 15, 822–829.
The histone methyltransferase Wolf–Hirschhorn syndrome candidate 1-like 1 (WHSC1L1) is involved in human carcinogenesis
Kang D., Cho HS., Toyokawa G., Kogure M., Yamane Y., Iwai Y., Hayami S., Tsunoda T., Field HI., Matsuda K., Neal DE., Ponder BA., Maehara Y., Nakamura Y., Hamamoto R.
Genes Chromosomes Cancer 2013, 52, 126-139.
Comprehensive Profiling of 8p11-12 Amplification in Breast Cancer
Gelsi-Boyer V., Orsetti B., Cervera N., Finetti P., Sircoulomb F., Rougé C., Lasorsa L., Letessier A., Ginestier C., Monville F., Esteyriès S., Adélaïde J., Esterni B., Henry C., Ethier SP., Bibeau F., Mozziconacci MJ., Charafe-Jauffret E., Jacquemier J., Bertucci F., Birnbaum D., Theillet C., Chaffanet M.
Molecular Cancer Research 2005, 3, 655.
When you plan a publication, please use the following acknowledgement:
BI-9321 was kindly provided by Boehringer Ingelheim via its open innovation platform opnMe, available at https://www.opnme.com.