SGC-STK17B-1 A chemical probe for STK17B/DRAK2 kinase

Probe and control are available from Sigma.

For any inquiries please contact proberequests@thesgc.org.

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Overview

SGC-STK17B-1

SGC-STK17B-1N (negative control)

Chemical structures of SGC-STK17B-1 (thieno[3,2-d]pyrimidine) and the negative control SGC-STK17B-1N (thieno[2,3-d]pyrimidine).

In a collaboration with Pfizer, the SGC has developed a high quality chemical probe and its negative control for protein kinase STK17B/DRAK2, Figure 1, which is a serine/threonine kinase part of the larger Death-associated protein kinase (DAPK) family that also includes DAPK1, DAPK2, DAPK3, and DRAK1. STK17B kinase is predominantly expressed in T-cells and B-cells. Due to this expression it has been identified as a possible therapeutic target for Type 1 diabetes, Multiple sclerosis and graft rejection. STK17B knock out mice are viable and show no abnormalities, but are remarkably resistant to autoimmune challenge. When crossed onto the NOD1 strain, a model of Type 1 diabetes1 or challenged with EAE, a model of Multiple Sclerosis, the STK17B knock out mice are protected from disease progression.2 STK17B is also overexpressed in hepatocellular3 and breast cancer.4 Silencing of STK17B in cells suggests that it may have utility in treatment of these cancers. The starting point in the development of this chemical probe was a series of thienopyrimidines donated by Pfizer, prepared in an effort targeting TPL2 kinase.5 From here, the SGC developed a library of small molecules inhibitors around the thieno[3,2-d]pyrimidine core optimizing for potency, selectivity, and cell activity and from this set, we identified SGC-STK17B-1 as a small molecule chemical probe.

Figure 1. Phylogenetic kinase tree, STK17B/DRAK2 and ST17A/DRAK1 highlighted with red circles. Illustration is reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).

Biological activity summary

Selectivity
SGC-STK17B-1 was profiled in the KINOMEscan assay against of 403 wild-type kinases @ 1 mM, followed by K_d determination of potential off-targets at Eurofins. SGC-STK17B-1 shown >30 fold selective (K_d) against the closest off targets STK17A/DRAK1, AURKB and CaMKK2. SGC-STK17B-1 is non-toxic in cells (HEK293 cells) in a time and dose dependent manner and have little to no effect on cell viability.

Cell-based assay
In cellular target engagement assays (NanoBRET) in HEK293 cells, SGC-STK17B-1 showed IC₅₀ of 190 nM against STK17B (n = 9) and >10,000 nM against DRAK1 and AURKB (potential off-targets). The negative control SGC-STK17B-1N shown IC₅₀ > 10,000 nM against STK17B.

Binding Activity
In a binding assay performed at Luceome Biotechnologies SGC-STK17B-1 showed potency of 43 nM against STK17B.

Acknowledgments. NanoBRET cellular assays were performed by Carrow Wells and Julie Pickett at SGC-UNC Chapel Hill.

Properties

Physical and chemical properties for SGC-STK17B-1
Molecular weight	358.46
Molecular formula	C16H10N2O2S3
IUPAC name	2-((6-(benzo[b]thiophen-2-yl)thieno[3,2-d]pyrimidin-4-yl)thio)acetic acid
MollogP	4.00
PSA	144.86
No. of chiral centres	0
No. of rotatable bonds	4
No. of hydrogen bond acceptors	4
No. of hydrogen bond donors	1
Storage	Stable as a solid at room temperature. DMSO stock solutions (up to 10mM) are stable at -20 °C
Dissolution	Soluble in DMSO up to 10 mM.

Physical and chemical properties for SGC-STK17B-1N
Molecular weight	358.46
Molecular formula	C16H10N2O2S3
IUPAC name	2-((6-(benzo[b]thiophen-2-yl)thieno[2,3-d]pyrimidin-4-yl)thio)acetic acid
MollogP	4.09
PSA	144.86
No. of chiral centres	0
No. of rotatable bonds	4
No. of hydrogen bond acceptors	4
No. of hydrogen bond donors	1
Storage	Stable as a solid at room temperature. DMSO stock solutions (up to 10mM) are stable at -20 °C.
Dissolution	Soluble in DMSO up to 10 mM.

SMILES:
SGC-STK17B-1: O=C(O)CSC1=C2C(C=C(C3=CC(C=CC=C4)=C4S3)S2)=NC=N1
SGC-STK17B-1N: O=C(O)CSC1=C2C(SC(C3=CC(C=CC=C4)=C4S3)=C2)=NC=N1

InChI:
SGC-STK17B-1: 1S/C16H10N2O2S3/c19-14(20)7-21-16-15-10(17-8-18-16)6-13(23-15)12-5-9-3-1-2-4-11(9)22-12/h1-6,8H,7H2,(H,19,20)
SGC-STK17B-1N: 1S/C16H10N2O2S3/c19-14(20)7-21-15-10-6-13(23-16(10)18-8-17-15)12-5-9-3-1-2-4-11(9)22-12/h1-6,8H,7H2,(H,19,20)

InChIKey:
SGC-STK17B-1: BNYXRPMABDQJJR-UHFFFAOYSA-N
SGC-STK17B-1N: RRIRDIKTUQVZCW-UHFFFAOYSA-N

Selectivity Profile

The KINOMEscan of SGC-STK17B-1 at 1mM showed few off-targets, Figure 1. With cutoff 10% control (90% inhibition), MET, NEK6, PIM2, WEE1, and CAMKK2 are shown in red circles in the TREEspot. This screening was followed by K_d determination and NanoBRET assays for several potential off-targets (Table 1).

Figure 1: Kinome selectivity and TREEspot analysis of 403 wild-type kinases for SGC-STK17B-1.

Results in Table 1 confirmed the high selectivity of SGC-STK17B-1 towards STK17B/DRAK2. Non-off targets were found for this chemical probe at concentrations shown.

Entry	Kinase	% Control (1 µM)	Enzyme assay K_d (nM)	Cell assay (NanoBRET) IC₅₀ (nM)
1	MET	0	>10,000	n.t
2	NEK6	0	>10,000	n.t
3	PIM2	0	nt	n.t
4	WEE1	0	>10,000	n.t
5	STK17B	0.9	34	190
6	CAMKK2	7.8	>10,000	n.t
7	AURKB	14	8947	>10,000
8	CAMKK1	14	5183	n.t
9	STK38L	29	>10,000	>10,000
10	CDKL1	40	>10,000	n.t
11	HIPK1	41	>10,000	>10,000
12	HIPK4	41	4352	>10,000
13	HIPK2	53	8612	>10,000
14	STK17A	73	4666	>10,000
15	STK16	77	648	n.t

Co-crystal structures

The co-crystal structure shown an unusual flip of R41 (P-loop) forming salt bridge network with inhibitor COOH group, R41 and E117 in one of the two experimentally observed conformations is a likely mechanism of selectivity. PDB depositions for the chemical probe are 6Y6F and 6Y6H (links will be updated as soon as they become available).

Important features:

Type-I binding mode
Back pocket interaction of COOH with lysine (K62)
Additional ethylene diol fills back pocket

Acknowledgments. Co-crystal structure was obtained by Apirat Chaikuad and Stefan Knapp at SGC-Frankfurt.

References

Farag, A.K. and E.J. Roh, Death-associated protein kinase (DAPK) family modulators: Current and future therapeutic outcomes. Med Res Rev, 2019. 39(1): p. 349-385.DOI: 10.1002/med.21518. https://www.ncbi.nlm.nih.gov/pubmed/29949198
Lan, Y., et al., STK17B promotes carcinogenesis and metastasis via AKT/GSK-3beta/Snail signaling in hepatocellular carcinoma. Cell Death Dis, 2018. 9(2): p. 236.DOI: 10.1038/s41419-018-0262-1. https://www.ncbi.nlm.nih.gov/pubmed/29445189
McGargill, M.A., et al., Drak2 regulates the survival of activated T cells and is required for organ-specific autoimmune disease. J Immunol, 2008. 181(11): p. 7593-605.DOI: 10.4049/jimmunol.181.11.7593. https://www.ncbi.nlm.nih.gov/pubmed/19017948
Ni, Y., et al., Identification and SAR of a new series of thieno[3,2-d]pyrimidines as Tpl2 kinase inhibitors. Bioorg Med Chem Lett, 2011. 21(19): p. 5952-6.DOI: 10.1016/j.bmcl.2011.07.069. https://www.ncbi.nlm.nih.gov/pubmed/21862328
Yang, K.M., et al., DRAK2 participates in a negative feedback loop to control TGF-beta/Smads signaling by binding to type I TGF-beta receptor. Cell Rep, 2012. 2(5): p. 1286-99.DOI: 10.1016/j.celrep.2012.09.028. https://www.ncbi.nlm.nih.gov/pubmed/23122956
Picado et al. A Chemical Probe for Dark Kinase STK17B Derives Its Potency and High Selectivity through a Unique P-Loop Conformation. J Med Chem 2020. 63 (23): p14626. https://doi.org/10.1021/acs.jmedchem.0c01174. https://pubs.acs.org/doi/....