SGC-GSK3-1 A chemical probe for GSK3.

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Overview

From a library of AT-7519 analogs, we identified a potent and cell-active chemical probe (SGC-GSK3-1) that inhibits glycogen synthase kinase-3 (GSK3⍺ and β). Comprehensive evaluation of kinome-wide selectivity confirmed that this GSK3 probe demonstrates remarkable selectivity. A structurally similar analog (SGC-CDKL5/GSK3-1N) was characterized as a negative control that does not bind to GSK3⍺ or GSK3β in corresponding cellular target engagement assays. At nanomolar concentrations, our GSK3 chemical probe promoted motor neuron survival when iPSC-derived motor neurons were exposed to ER stress. When used at an appropriate concentration (<500 nM) in cells, SGC-GSK3-1 is exquisitely selective for GSK3⍺ or GSK3β. Our chemical probe set can be used alongside other potent and selective, structurally divergent GSK3 inhibitors to characterize the impacts of GSK3 inhibition on downstream biology.

Properties

Physical and chemical properties for SGC-GSK3-1
Molecular weight	364.35
Molecular formula	C₁₇H₁₈F₂N₄O₃
IUPAC name	4-(2,6-difluorobenzamido)-N-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazole-3-carboxamide
ClogP	-0.52
PSA	96.11
No. of chiral centers	0
No. of rotatable bonds	7
No. of hydrogen bond acceptors	6
No. of hydrogen bond donors	3
Storage	Stable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20^oC
Dissolution	Soluble in DMSO up to 10 mM

Physical and chemical properties for SGC-CDKL5/GSK3-1N
Molecular weight	363.37
Molecular formula	C₁₇H₁₉FN₅O₂
IUPAC name	4-(3,5-difluorobenzamido)-1-methyl-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide
ClogP	-0.32
PSA	88.05
No. of chiral centers	0
No. of rotatable bonds	6
No. of hydrogen bond acceptors	6
No. of hydrogen bond donors	3
Storage	Stable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20^oC
Dissolution	Soluble in DMSO up to 10 mM

SMILES:

SGC-GSK3-1: O=C(C1=NNC=C1NC(C2=C(F)C=CC=C2F)=O)NCC3CCOCC3

SGC-CDKL5/GSK3-1N: O=C(C1=NN(C)C=C1NC(C2=CC(F)=CC(F)=C2)=O)NC3CCNCC3

InChI:

SGC-GSK3-1: InChI=1S/C17H18F2N4O3/c18-11-2-1-3-12(19)14(11)16(24)22-13-9-21-23-15(13)17(25)20-8-10-4-6-26-7-5-10/h1-3,9-10H,4-8H2,(H,20,25)(H,21,23)(H,22,24)

SGC-CDKL5/GSK3-1N: InChI=1S/C17H19F2N5O2/c1-24-9-14(22-16(25)10-6-11(18)8-12(19)7-10)15(23-24)17(26)21-13-2-4-20-5-3-13/h6-9,13,20H,2-5H2,1H3,(H,21,26)(H,22,25)

InChIKey:

SGC-GSK3-1: OCQCKUTZHBQNEI-UHFFFAOYSA-N

SGC-CDKL5/GSK3-1N: ODZBDODDFLVDQZ-UHFFFAOYSA-N

Selectivity Profile

SGC-GSK3-1 was profiled in the DiscoverX scanMAX assay against 403 wild-type kinases at 1 μM. Only 5 kinases showed PoC <10 giving an S₁₀(1 μM) = 0.012. When the PoC <35 fraction was examined, 15 kinases were included (S₃₅(1 μM) = 0.037). Potential off-targets within the S₃₅(1 μM) fraction were tested via biochemical enzymatic/binding assays plus NanoBRET target engagement assays for CDKL5, GSK3⍺, GSK3β, and DYRK1B. Data corresponding with off-target kinase activity is shown in the table below.

Figure 2: SGC-GSK3-1 was profiled in the DiscoverX scanMAX assay against 403 wild-type kinases at 1 μM and off-target kinases inhibited PoC <35 were tested in an orthogonal assay. Rows colored green are GSK3⍺, GSK3β, and DYRK1B. No other kinases demonstrate enzymatic IC₅₀ values within 30-fold of the GSK3β enzymatic IC₅₀ value.

In Vitro Potency

Figure 1: Kinome tree with GSK3⍺ and GSK3β highlighted as red circles. Illustration is reproduced courtesy of Eurofins DiscoverX (http://treespot.discoverx.com).

Biological activity summary:

Enzymatic assay (Eurofins): GSK3⍺ IC₅₀ = 1.0 nM and GSK3β IC₅₀ = 2.0 nM
Cellular data (NanoBRET): GSK3⍺ IC₅₀ = 4.6 nM and GSK3β IC₅₀ = 12 nM
Only 5/403 kinases with PoC <10 when screened at 1 μM

Cell-based Assay Data

A NanoBRET assay was utilized to assess the binding affinity of SGC-GSK3-1 to CDKL5, GSK3⍺, GSK3β, and DYRK1B. The negative control shows no binding affinity for GSK3⍺ or GSK3β.

Figure 3: SGC-GSK3-1 was profiled in the GSK3⍺ and GSK3β NanoBRET assays.

Figure 4: SGC-CDKL5/GSK3-1N was profiled in the GSK3⍺ and GSK3β NanoBRET assays.

References

References

Ong, H. W.; Liang, Y.; Richardson, W.; Lowry, E. R.; Wells, C. I.; Chen, X.; Silvestre, M.; Dempster, K.; Silvaroli, J. A.; Smith, J. L.; Wichterle, H.; Pabla, N. S.; Ultanir, S. K.; Bullock, A. N.; Drewry, D. H.*; Axtman, A. D.* Discovery of a potent and selective CDKL5/GSK3 chemical probe that is neuroprotective. ACS Chem Neurosci 2023, 14, 1672–1685; 10.1021/acschemneuro.3c00135.

Ong, H. W.; Liang, Y.; Richardson, W.; Lowry, E. R.; Wells, C. I.; Chen, X.; Silvestre, M.; Dempster, K.; Silvaroli, J. A.; Smith, J. L.; Wichterle, H.; Pabla, N. S.; Ultanir, S. K.; Bullock, A. N.; Drewry, D. H.*; Axtman, A. D.* A potent and selective CDKL5/GSK3 chemical probe is neuroprotective. BioRxiv 2023, doi: 10.1101/2023.02.09.527935.