NSD3-PWWP1 Antagonist | BI-9321
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Highlights
BI-9321 was discovered in collaboration with the Structural Genomics Consortium (SGC). It represents a potent, selective and cellular active antagonist of the NSD3-PWWP1 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. Treatment of MOLM-13 cells with BI-9321 results into the downregulation of Myc mRNA in cells and reduced proliferation which cannot be observed with the available negative control BI-9466.
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Target 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.
BI-9321 in complex with NDS3
In vitro Activity
|
Probe name / Negative control |
BI-9321 |
BI-9466 |
|
MW [Da] |
360.4 |
295.4 |
|
MW [Da] HCl salt (BI-9321 will be delivered as the HCl salt) |
396.9 |
331.9 |
|
TR-FRETa (IC50) [nM] |
203 |
102 000 |
|
SPR (Kd)b [nM] |
166 |
144 000 |
|
ITC (Kd)c [nM] |
445 |
n.d. |
a 1x PBS; 0.05% Tween20; 0.1 % BSA; filtered
b 50 mM TRIS, pH 8.0; 150 mM NaCl; 1 mM TCEP; 0.005 % Tween 20; 2% DMSO
c 20 mM HEPES, 100 mM NaCl, 3% DMSO, pH 8.0
In vitro DMPK and CMC parameters
|
Probe name / Negative control |
BI-9321 |
BI-9466 |
|
logP |
n.d. |
n.d. |
|
Solubility @ pH 6.8 [µg/ml] |
> 100 |
n.d. |
|
CACO permeability @ pH7.4 [*10-6 cm/s] |
16 |
n.d. |
|
CACO 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. |
Negative control
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-9321 which serves as a negative control
Selectivity
BI-9321 did not hit any of the 31 tested kinases.
|
BI-9321 |
Selectivity data available |
|
Cerep® |
No |
|
Panlabs® |
No |
|
Invitrogen® |
Yes |
|
DiscoverX® |
No |
|
Dundee |
No |
Co-crystal structure of the BI probe compound and the target protein
The X-ray crystal structure of NSD3-PWWP1 in complex with BI-9321 is available (PDB code: 6G2O)1.
Summary
BI-9321 is a potent and highly selective antagonist of the PWWP1 domain of NSD3. It is a first in class chemical probe, targeting the methyl-lysine binding site, developed in collaboration with the Structural Genomics Consortium (SGC). BI-9321 exhibits an in vitro potency of 200 nM and cellular target engagement at around 1 µM. In MOLM-13 cells BI-9321 downregulates Myc mRNA and impairs proliferation. A closely-related compound, BI-9466 is 500-fold less active (TR-FRET assay) and is a recommended negative control. Both compounds should be used in parallel in a dose response range between 0.1 and 20µM.
Supplementary data
References
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.
NSD3-short is an adaptor protein that couples BRD4 to the CHD8 chromatin remodeler
Shen C., Ipsaro JJ., Shi J., Milazzo JP., Wang E., Roe JS., Suzuki Y., Pappin DJ., Joshua-Tor L., Vakoc CR.
Molecular cell 2015, 60, 847-859.
Structural and Histone Binding Ability Characterizations of Human PWWP Domains
Wu H., Zeng H., Lam R., Tempel W., Amaya MF., Xu C., Dombrovski L., Qiu W., Wang Y., Min J.
PLoS ONE 2011, 6, e18919.
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.
