SGC-CAF382-1 A chemical probe for CDKL5.

The probe may be purchased from Tocris ans Sigma.

overview
properties
selectivity profile
in vitro potency
cell based assay data
references

overview

Probe		Negative control

SGC-CAF382-1		SGC-CAF268-1N

From a library of SNS-032 analogs, we identified a potent and cell-active chemical probe (SGC-CAF382-1) that inhibits cyclin-dependent kinase-like 5 (CDKL5) and several cyclin-dependent kinases (CDK9, CDK16, CDK17, and CDK18). Comprehensive evaluation of kinome-wide selectivity confirmed that this chemical probe demonstrates good selectivity. A structurally similar analog (SGC-CAF268-1N) was characterized as a negative control that does not bind to CDKL5, CDK9, CDK16, CDK17, or CDK18 in corresponding cellular target engagement assays. SGC-CAF382-1 is devoid of GSK3 inhibition when used at an appropriate concentration. At nanomolar concentrations, our chemical probe promoted motor neuron survival when iPSC-derived motor neurons were subjected to ER stress. An orthogonal CDKL5 chemical probe, SGC-CDKL5/GSK3-1, represents a distinct chemotype with non-overlapping kinase off-targets that can be profiled in parallel. When used at ≤100 nM in cell-based assays, our chemical probe set can help the community further characterize the underexplored roles of CDKL5.

properties

Probe		Negative control

SGC-CAF382-1		SGC-CAF268-1N

Physical and chemical properties for SGC-CAF382-1
Molecular weight	366.50
Molecular formula	C₁₆H₂₂N₄O₂S₂
IUPAC name	N-(5-(((5-isopropyloxazol-2-yl)methyl)thio)thiazol-2-yl)piperidine-4-carboxamide
ClogP	0.16
PSA	133.59
No. of chiral centers	0
No. of rotatable bonds	7
No. of hydrogen bond acceptors	5
No. of hydrogen bond donors	2
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-CAF268-1N
Molecular weight	493.57
Molecular formula	C₂₂H₂₂F₃N₅OS₂
IUPAC name	2-(4-methylpiperazin-1-yl)-N-(5-((3-(trifluoromethyl)benzyl)thio)thiazol-2-yl)isonicotinamide
ClogP	2.33
PSA	114.90
No. of chiral centers	0
No. of rotatable bonds	8
No. of hydrogen bond acceptors	7
No. of hydrogen bond donors	1
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-CAF382-1: O=C(C1CCNCC1)NC2=NC=C(SCC3=NC=C(C(C)C)O3)S2

SGC-CAF268-1N: O=C(NC1=NC=C(SCC2=CC=CC(C(F)(F)F)=C2)S1)C3=CC=NC(N4CCN(C)CC4)=C3

InChI:

SGC-CAF382-1: InChI=1S/C16H22N4O2S2/c1-10(2)12-7-18-13(22-12)9-23-14-8-19-16(24-14)20-15(21)11-3-5-17-6-4-11/h7-8,10-11,17H,3-6,9H2,1-2H3,(H,19,20,21)

SGC-CAF268-1N: InChI=1S/C22H22F3N5OS2/c1-29-7-9-30(10-8-29)18-12-16(5-6-26-18)20(31)28-21-27-13-19(33-21)32-14-15-3-2-4-17(11-15)22(23,24)25/h2-6,11-13H,7-10,14H2,1H3,(H,27,28,31)

InChIKey:

SGC-CAF382-1: MCLDWKVRXDHDEI-UHFFFAOYSA-N

SGC-CAF268-1N: RUYKDMMLESXEOA-UHFFFAOYSA-N

selectivity profile

SGC-CAF382-1 was profiled in the DiscoverX scanMAX assay against 403 wild-type kinases at 1 μM. Only 7 kinases showed PoC <10 giving an S₁₀(1 μM) = 0.017. When the PoC <35 fraction was examined, 10 kinases were included (S₃₅(1 μM) = 0.024). Potential off-targets within the S₃₅(1 μM) fraction and GSK3β (PoC = 51) were tested via biochemical enzymatic/binding assays plus NanoBRET target engagement assays for CDKL5, GSK3⍺, GSK3β, CDK9, CDK16, CDK17, and CDK18. Data corresponding with off-target kinase activity is shown in the table below.

SGC-CAF268-1N was also tested in the DiscoverX scanMAX panel and 2 kinases had a PoC <35 (S₃₅(1 μM) = 0.005). The negative control was evaluated in the corresponding NanoBRET assay for DYRK1B, the kinase that bound with the lowest PoC value in the kinome-wide screen. All results are in the table below.

in vitro potency

Biological activity summary:

Binding assay (Luceome): CDKL5 IC₅₀ = 6.7 nM; Enzymatic assay (Eurofins): CDK9 IC₅₀ = 20 nM, CDK16 IC₅₀ = 62 nM, CDK17 IC₅₀ = 89 nM, CDK18 IC₅₀ = 100 nM
Cellular data (NanoBRET): CDKL5 IC₅₀ = 10 nM; CDK9 IC₅₀ = 280 nM, CDK16 IC₅₀ = 390 nM, CDK17 IC₅₀ = 240 nM, CDK18 IC₅₀ = 260 nM
Only 7/403 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-CAF382-1 to CDKL5, CDK9, CDK16, CDK17, CDK18, GSK3⍺, and GSK3β. The negative control shows no binding affinity for CDKL5, CDK9, CDK16, CDK17, or CDK18.

references

Castano, A.; Silvestre, M.; Wells, C. I.; Sanderson, J. L.; Ferrer, C. A.; Ong, H. W.; Liang, Y.; Richardson, W.; Silvaroli, J. A.; Bashore, F. M.; Smith, J. L.; Genereux, I. M.; Dempster, K.; Drewry, D. H.; Pabla, N. S.; Bullock, A. N.; Benke, T. A.; Ultanir, S.; Axtman, A. D. Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology. Elife 2023, 12, e88206; doi: 10.7554/eLife.88206.

pk properties

co-crystal structures

synthetic schemes

materials and methods

SGC-CDKL5/GSK3 A dual activity chemical probe for CDKL5 and GSK3.

This probe is available from Sigma.

overview
properties
selectivity profile
in vitro potency
cell based assay data
references

overview

Probe		Negative control

SGC-CDKL5/GSK3-1		SGC-CDKL5/GSK3-1N

From a library of AT-7519 analogs, we identified a potent and cell-active chemical probe (SGC-CDKL5/GSK3-1) that inhibits cyclin-dependent kinase-like 5 (CDKL5) and glycogen synthase kinase-3 (GSK3⍺ and β). Comprehensive evaluation of kinome-wide selectivity confirmed that this CDKL5/GSK3 probe demonstrates exceptional selectivity. A structurally similar analog (SGC-CDKL5/GSK3-1N) was characterized as a negative control that does not bind to CDKL5, GSK3⍺, or GSK3β in corresponding cellular target engagement assays. At nanomolar concentrations, our CDKL5/GSK3 chemical probe promoted motor neuron survival when iPSC-derived motor neurons were subjected to ER stress. We recommend use of a structurally related GSK3 chemical probe, SGC-GSK3-1, in tandem to interrogate those effects due to GSK3 rather than CDKL5 inhibition. An orthogonal CDKL5 chemical probe, SGC-CAF382-1, represents a distinct chemotype with non-overlapping kinase off-targets that can also be used in parallel. Our chemical probe set can be used by the community to further characterize the underexplored roles of CDKL5 and how inhibition of CDKL5 and GSK3 impacts downstream biology.

properties

Probe		Negative control

SGC-CDKL5/GSK3-1		SGC-CDKL5/GSK3-1N

Physical and chemical properties for SGC-CDKL5/GSK3-1
Molecular weight	349.34
Molecular formula	C₁₆H₁₇FN₅O₂
IUPAC name	4-(3,5-difluorobenzamido)-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide
ClogP	-0.33
PSA	98.91
No. of chiral centers	0
No. of rotatable bonds	6
No. of hydrogen bond acceptors	6
No. of hydrogen bond donors	4
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-CDKL5/GSK3-1: O=C(NC1CCNCC1)C2=NNC=C2NC(C3=CC(F)=CC(F)=C3)=O

SGC-CDKL5/GSK3-1N: O=C(NC1CCNCC1)C2=NN(C=C2NC(C3=CC(F)=CC(F)=C3)=O)C

InChI:

SGC-CDKL5/GSK3-1: InChI=1S/C16H17F2N5O2/c17-10-5-9(6-11(18)7-10)15(24)22-13-8-20-23-14(13)16(25)21-12-1-3-19-4-2-12/h5-8,12,19H,1-4H2,(H,20,23)(H,21,25)(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-CDKL5/GSK3-1: NIHAFOURWLZLFN-UHFFFAOYSA-N

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

selectivity profile

SGC-CDKL5/GSK3-1 was profiled in the DiscoverX scanMAX assay against 403 wild-type kinases at 1 μM. Only 4 kinases showed PoC <10 giving an S₁₀(1 μM) = 0.01. When the PoC <35 fraction was examined, 11 kinases were included (S₃₅(1 μM) = 0.027). 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, CDK16, and CDK17. Data corresponding with off-target kinase activity is shown in the table below.

Figure 2: SGC-CDKL5/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 CDKL5, GSK3⍺, and GSK3β. No other kinases demonstrate enzymatic IC₅₀ values within 30-fold of the GSK3β enzymatic IC₅₀ value.

in vitro potency

Figure 1: Kinome tree with CDKL5, GSK3⍺ and GSK3β highlighted as red circles. Illustration is reproduced courtesy of Eurofins DiscoverX (http://treespot.discoverx.com).

Biological activity summary:

Binding assay (Luceome): CDKL5 IC₅₀ = 6.5 nM; Enzymatic assay (Eurofins): GSK3⍺ IC₅₀ = 4.0 nM and GSK3β IC₅₀ = 9.0 nM
Cellular data (NanoBRET): CDKL5 IC₅₀ = 3.5 nM; GSK3⍺ IC₅₀ = 10 nM; GSK3β IC₅₀ = 35 nM
Only 4/403 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-CDKL5/GSK3-1 to CDKL5, GSK3⍺, GSK3β, CDK16, and CDK17. The negative control shows no binding affinity for CDKL5, GSK3⍺, or GSK3β.

Figure 3: SGC-CDKL5/GSK3-1 was profiled in the CDKL5, GSK3⍺, and GSK3β NanoBRET assays.

Figure 4: SGC-CDKL5/GSK3-1N was profiled in the CDKL5, 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.

pk properties

co-crystal structures

synthetic schemes

materials and methods

SGC-GSK3-1 A chemical probe for GSK3.

This probe is available from Sigma.

overview
properties
selectivity profile
in vitro potency
cell based assay data
references

overview

Probe		Negative control

SGC-GSK3-1		SGC-CDKL5/GSK3-1N

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

Probe		Negative control

SGC-GSK3-1		SGC-CDKL5/GSK3-1N

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(NCC1CCOCC1)C2=NNC=C2NC(C3=C(C=CC=C3F)F)=O

SGC-CDKL5/GSK3-1N: O=C(NC1CCNCC1)C2=NN(C=C2NC(C3=CC(F)=CC(F)=C3)=O)C

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.* A potent and selective CDKL5/GSK3 chemical probe is neuroprotective. BioRxiv 2023, doi: 10.1101/2023.02.09.527935.

pk properties

co-crystal structures

synthetic schemes

materials and methods

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Unleashing the Power of AI in Drug Design: Paving the Path to Open Chemistry Data

by: SGC

The field of drug discovery has continuously evolved over time, driven by advancements in scientific understanding and technological innovations. From serendipitous discoveries, such as the discovery of aspirin and penicillin, to targeted pharmacology and high-throughput screenings, the process of identifying new therapeutic agents has undergone significant transformations.

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