Probe		Negative control
SKI-73		SKI-73N

The SGC in collaboration with the Memorial Sloan Kettering Cancer Center has developed SKI-73, a chemical probe for PRMT4.  SKI-73 is active in cells and is the prodrug of SKI-72, a potent and selective inhibitor of PRMT4.

Data relating to the discovery of this probe is being prepared for publication.  In the meantime, in order to facilitate research by the community we are making this compound available through this website

Probe		Negative control
BI-9321		BI-9466

A collaboration between Boehringer Ingelheim and the SGC has resulted in the discovery of BI-9321, a potent and selective antagonist of the PWWP1 domain of NSD3. BI-9321 binds to the PWWP1 domain of NSD3 with a K_d of 166 nM by SPR and is selective over other PWWP domains including NSD2-PWWP1 and NSD3-PWWP2. BI-9321 antagonizes the interaction of H3 with NSD3-PWWP1 in U2OS cells as measured by NanoBRET with IC₅₀ of 1.2 µM.  A closely-related compound, BI-9466 is 200-fold less active and is a recommended negative control. Both compounds should be used in parallel in a dose response range between 0.1 and 20µM.

Data relating to the discovery of this probe is being prepared for publication. In the meantime, in order to facilitate research by the community we are making this compound available through this website.

Probe		Negative control
MRK-740		MRK-740-NC

A collaboration between MSD and the SGC has resulted in the discovery of MRK-740, a potent inhibitor of PRDM9 with a peptide competitive MOA. MRK-740 inhibits in vitro methylation of H3K4 with IC50 = 85 nM and shows more than 100-fold selectivity over other histone methyltransferases and other non-epigenetic targets. MRK-740 inhibits the methylation of H3K4 in cells with IC50 = 0.8 µM.  A control compound, MRK-740-NC, has also been developed which inhibits the in vitro methylation of H3K4 with IC50 > 100 µM. The use of this compound at 3 µM is recommended in cells.

Data relating to the discovery of this probe is being prepared for publication. In the meantime, in order to facilitate research by the community we are making this compound available through this website.

Probe		Negative control
SGC3027 (Pro-drug of SGC8158)		SGC3027N (Pro-drug of SGC8158N)
Active component		Negative control
SGC8158		SGC8158N

A collaboration between SGC, Takeda, and OICR has resulted in the discovery of SGC3027, the first potent, selective and cell active chemical probe for PRMT7. SGC3027 is a pro-drug of SGC8158 which releases the active component upon reduction in the cell by reductases.  The in vitro activity of SGC8158, the active component, includes inhibition of PRMT7 with IC50  < 2.5 nM for methylation of H2B (23-37) and greater than 40-fold selectivity over other histone methyltransferases and non-epigenetic targets. In cellular assays using C2C12 cells, SGC3027 inhibits the methylation of HSP70 with IC50 = 2.4 microM.

A closely related compound, SGC3027N, is significantly less active in the cellular assay, and is an ideal control compound for cellular studies.

Data relating to the discovery of this probe is being prepared for publication.  In the meantime, in order to facilitate research by the community we are making this compound available through this website

Probe		Negative control
SGC-AAK1-1		SGC-AAK1-1N

AAK1 (adaptor protein 2-associated kinase) and BMP2K/BIKE (BMP-2 inducible kinase) comprise half of the numb-associated kinase (NAK) family, which also includes cyclin G associated kinase (GAK) and STK16/MPSK1 (serine/threonine kinase 16/myristoylated and palmitoylated serine/threonine kinase 1).¹

AAK1 is a 104 kDa serine/threonine kinase with broad tissue expression. Within the cell AAK1 localizes to the cell membrane and cytoplasm.² AAK1 is involved in clathrin-mediated endocytosis (CME), both by direct binding to clathrin and by phosphorylation of the medium subunit of AP-2 (adaptor protein 2).^3-5 In this manner, AAK1 has been identified as a negative regulator of Wnt signaling via mediation of LRP6 internalization. Studies have implicated multiple roles for AAK1 in influencing Notch signaling, including priming and redistribution of Numb as well as Notch activation.^6-7

Relatively less is known about the highly understudied kinase BIKE. BIKE is broadly expressed, and in the cell, it localizes to nuclear speckles.² BIKE was originally identified as its expression was observed to increase upon bone morphogenic protein (BMP-2)-induced differentiation of a prechondroblastic cell line.⁸ The same study provided evidence for BIKE having an important regulatory role in attenuating the program of osteoblast differentiation. Proteomic studies identified BIKE as a clathrin vesicle-associated protein and have also identified interaction between BIKE and Numb.^9-10

NAK family domain structures

Location of AAK1 and BIKE on kinome tree

Snapshot of crystal structure of acylaminoindazole bound to BIKE

SGC-AAK1-1 is a chemical probe for AAK1 and BIKE that potently targets the ATP-binding site (AAK1 K_i =  9.1 nM; BIKE K_i = 17 nM). Regarding kinase selectivity, only three kinases were observed to bind SGC-AAK1-1 within 30-fold of the K_D of AAK1 in a KINOMEscan assay (DiscoverX) at 1 μM followed by K_D determinations: RIOK1 (K_D = 72 nM), RIOK3 (K_D = 290 nM), and PIP5K1C (K_D = 260 nM). In a live cell NanoBRET assay (Promega) SGC-AAK1-1 has potency for ectopically expressed full-length AAK1- and BIKE-Nluc fusion proteins (AAK1 IC₅₀ = 230 nM; BIKE IC₅₀ = 1.5 μM).

A chemically related negative control compound, SGC-AAK1-1N, is provided.