Probe		Negative control
DDR-TRK-1		DDR-TRK-1N

The discoidin domain receptors (DDRs), DDR1 and DDR2, are unique among the receptor tyrosine kinases (RTKs) in being activated by interaction with the extracellular matrix via binding to triple-helical collagen by the receptor extracellular domains.¹  DDR1 and DDR2 form constitutive dimers making them unusual among RTKs, which typically dimerize only upon activation.² DDRs regulate extracellular matrix remodeling, as well as cell adhesion, proliferation and migration.³ DDR kinases are linked to the progression of various human diseases, including fibrotic disorders, atherosclerosis and cancer.^3-5  Significantly, they are identified as indicators of poor prognosis in ovarian, breast and lung cancer.⁶ DDR1 overexpression is associated with increased cell survival and invasion in hepatocellular carcinomas, pituitary adenoma and prostate cancer,⁷ whereas DDR2 is mutated in squamous cell lung cancers⁸ and contributes to breast cancer metastasis.⁹ The promise of DDR kinases as a therapeutic target has been demonstrated by DDR1 knockdown that has been shown to reduce metastatic activity in lung cancer models,¹⁰ slow the development of atherosclerosis,⁵ and impede the development of fibrotic disorders.¹¹

The TRK kinases are represented by three members, TRKA, TRKB, and TRKC, which are selectively expressed in neuronal tissue.  Receptor signaling is initiated by binding of the neurotrophic factors NGF, BDNF, and NTR, respectively.  Subsequent signaling is via the RAS, PLCγ and PI3γ pathways.  The TRKs play a vital role in CNS development and survival. Gene fusions, protein overexpression, and single nucleotide alterations, have been implicated in the pathogenesis of specific cancer types including glioblastoma, papillary thyroid carcinoma, and secretory breast carcinomas, but are rare in most other cancers.¹²

DDR-TRK-1 is a chemical probe for the DDR and TRK kinases with good in vitro and in vivo potency.
DDR-TRK-1 inhibits colony formation and migration of Panc-1 pancreatic cancer cells.  In cellular and mouse models of lung fibrosis, DDR-TRK-1 inhibits signaling, expression of fibrotic markers and fibrotic features such as hydroxproline expression.  With restricted CNS exposure and therefore no TRK inhibition, the in vivo effects of DDR-TRK-1 can be attributed to DDR1-2 inhibition. DDR-TRK-1N is the negative control compound with very minimal differences in compound structure, and should be used in parallel to DDR-TRK-1. The probe can be complemented by the use of BAY-826, which inhibits the kinases TIE1, TIE2, DDR1 and DDR2, but lacks TRK activity to better understand the target involved in the phenotypic effect.

Work on this probe has been published in J.Med.Chem. 2016 59(12), p 5911-5916, 'Structure-Based Design of Tetrahydroisoquinoline-7-carboxamides as Selective Discoidin Domain Receptor 1 (DDR1) inhibitors'.

Potency Against Target Family

DDR-TRK-1 is a chemical probe for the DDR and TRK kinases (IC₅₀ 3-43 nM) with corresponding good cellular potency in NanoBRET^TM target engagement assays (IC₅₀ 104-448 nM).  DDR-TRK-1 was shown to be selective in an in vitro kinase panel followed by cellular NanoBRET^TM assays.

Selectivity

DDR-TRK-1 is selective in KINOMEscan® at 1μM. The closest off target is CDK11 (370 nM), which was however shown to be only poorly inhibited in cells  (~5 µM in NanoBRET^TM). The negative control DDR-TRK-1N is entirely clean in KINOMEscan at 1μM.

Dosage

We recommend that DDR-TRK-1 be used at 5μM concentration in cells. The negative control DDR-TRK-1N should be used at 5μM concentration in cells – it is toxic in HeLa cells above 10μM.

We also recommend the use of selective TRK inhibitors or the TIE-DDR BAY-826 probe in parallel to dissect the biology of DDR1/2 versus TRKA/B/C.

Cellular Activity

In NanoBRET^TM assays, DDR-TRK-1 shows a potency of 104nM against DDR1, 175nM against DDR2, 448nM against TRKA and 142nM against TRKB.

In vitro Activity

In an activity assay at RBC (10μM ATP), DDR-TRK-1 shows an IC50 value of 27nM against DDR1, 4.5nM against DDR2, 43nM against TRKA, 3.6nM against TRKB and 2.9nM against TRKC.

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