29.09.2023

SGC 20th Anniversary Symposium Europe RECAP: From protein structures to probing biology

by: SGC

Twenty years have now passed since the inception of the Structural Genomics Consortium (SGC), marking a remarkable journey dedicated to advancing knowledge through open science and collaboration.

On Wednesday, September 6th, the SGC Karolinska hosted the 20th Anniversary Symposium Europe in Stockholm. This symposium served as a continuation of the celebration that began in Toronto earlier in March, commemorating past achievements and charting the course for what lies ahead.

28.09.2023

Structural Genomics Consortium and X-Chem Enter into Collaboration to Unlock the Human Proteome and Promote Open Science

by: SGC

Toronto, Canada, September 28, 2023 – The Structural Genomics Consortium (SGC), a public-private partnership dedicated to accelerating drug discovery through open science and X-Chem, a global leader in innovative drug discovery services, announced that they have entered a collaboration aimed at creating new chemical tools to study human proteins.

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 weight366.50
Molecular formulaC16H22N4O2S2
IUPAC nameN-(5-(((5-isopropyloxazol-2-yl)methyl)thio)thiazol-2-yl)piperidine-4-carboxamide
ClogP0.16
PSA133.59
No. of chiral centers0
No. of rotatable bonds7
No. of hydrogen bond acceptors5
No. of hydrogen bond donors2
StorageStable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20oC
DissolutionSoluble in DMSO up to 10 mM
Physical and chemical properties for SGC-CAF268-1N
Molecular weight493.57
Molecular formulaC22H22F3N5OS2
IUPAC name2-(4-methylpiperazin-1-yl)-N-(5-((3-(trifluoromethyl)benzyl)thio)thiazol-2-yl)isonicotinamide
ClogP2.33
PSA114.90
No. of chiral centers0
No. of rotatable bonds8
No. of hydrogen bond acceptors7
No. of hydrogen bond donors1
StorageStable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20oC
DissolutionSoluble 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.

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. BioRxiv 2023, doi: 10.1101/2023.04.24.538049.

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 weight349.34
Molecular formulaC16H17FN5O2
IUPAC name4-(3,5-difluorobenzamido)-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide
ClogP-0.33
PSA98.91
No. of chiral centers0
No. of rotatable bonds6
No. of hydrogen bond acceptors6
No. of hydrogen bond donors4
StorageStable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20oC
DissolutionSoluble in DMSO up to 10 mM
Physical and chemical properties for SGC-CDKL5/GSK3-1N
Molecular weight363.37
Molecular formulaC17H19FN5O2
IUPAC name4-(3,5-difluorobenzamido)-1-methyl-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide
ClogP-0.32
PSA88.05
No. of chiral centers0
No. of rotatable bonds6
No. of hydrogen bond acceptors6
No. of hydrogen bond donors3
StorageStable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20oC
DissolutionSoluble 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 weight364.35
Molecular formulaC17H18F2N4O3
IUPAC name4-(2,6-difluorobenzamido)-N-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazole-3-carboxamide
ClogP-0.52
PSA96.11
No. of chiral centers0
No. of rotatable bonds7
No. of hydrogen bond acceptors6
No. of hydrogen bond donors3
StorageStable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20oC
DissolutionSoluble in DMSO up to 10 mM
Physical and chemical properties for SGC-CDKL5/GSK3-1N
Molecular weight363.37
Molecular formulaC17H19FN5O2
IUPAC name4-(3,5-difluorobenzamido)-1-methyl-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide
ClogP-0.32
PSA88.05
No. of chiral centers0
No. of rotatable bonds6
No. of hydrogen bond acceptors6
No. of hydrogen bond donors3
StorageStable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20oC
DissolutionSoluble 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.* 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
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