The probe and control may be requested here.
| Probe | Negative control | |
 |
|  |
JA310 |
| JA262 |
Mammalian STE20-like protein kinase 3 and -4 (MST3/4) are part of the MST family, which comprises three additional members, namely MST1, MST2 and YSK1. The MSTs are involved in cell proliferation, cell migration and cell polarity.[1] For MST3 it has been shown that it can phosphorylate STK38L and stimulate its kinase activity [2]; for MST4 ATG4B has been demonstrated as a substrate and thereby regulates the autophagic activity.[3]
SGC has developed JA310, a potent and selective MST3 inhibitor with a EC50 determined by a NanoBret of 106 nM for MST3 in intact cells and 76 nM in permeabilized cells. JA310 shows kinome wide selectivity in a screening against 340 WT kinases (from Reaction Biology). In addition, the chemical probe (JA310) is accompanied by a negative control (JA262), which is structurally closely related to the probe molecule.[4]
Selectivity
JA310 has been shown to be selective in an in vitro kinase panel from Reaction Biology against 340 WT-Kinases followed by cellular NanoBRET assays.
Dosage
To distinguish the effect between MST3 and MST4 in a cell-based assays, we recommend to use JA310 together with the neg. control compound JA262 at a concentration of no higher than 1 µM for cell-based assays.
Cellular Activity
JA310 displayed an EC50 of 106 nM in the NanoBRETTM assay.
| Probe | Negative control | |
|
| |
JA310 |
| JA262 |
Physical and chemical properties JA310 | |
| MW | 337.39 |
| MF | C17H19N7O |
| IUPAC name | 2,4,5,8,14,16,23-heptaazatetracyclo[13.7.1.1³,⁶.0¹⁷,²²]tetracosa-1(22),3,6(24),15(23),16,18,20-heptaen-7-one |
| logP | 2.46 |
| PSA | 107.62 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 0 |
| No. of hydrogen bond acceptors | 6 |
| No. of hydrogen bond donors | 4 |
| Storage | stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | soluble in DMSO in a concentration of 10 mM |
SMILES: O=C(C1=CC(N2)=NN1)NCCCCCNC3=NC2=C4C=CC=CC4=N3
InChI: InChI=1S/C17H19N7O/c25-16-13-10-14(24-23-13)21-15-11-6-2-3-7-12(11)20-17(22-15)19-9-5-1-4-8-18-16/h2-3,6-7,10H,1,4-5,8-9H2,(H,18,25)(H3,19,20,21,22,23,24)
InChIKey: XBWKVZHZUZNMJE-UHFFFAOYSA-N
Physical and chemical properties JA262 | |
| MW | 323.74 |
| MF | C12H14ClN7O2 |
| IUPAC name | 18-chloro-11-oxa-2,4,5,8,14,16,19-heptaazatricyclo[13.3.1.1³,⁶]icosa-1(18),3,6(20),15(19),16-pentaen-7-one |
| logP | 0.46 |
| PSA | 116.85 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 0 |
| No. of hydrogen bond acceptors | 7 |
| No. of hydrogen bond donors | 4 |
| Storage | stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | soluble in DMSO in a concentration of 10 mM |
SMILES: O=C(C1=CC(N2)=NN1)NCCOCCNC3=NC2=C(C=N3)Cl
InChI: InChI=1S/C12H14ClN7O2/c13-7-6-16-12-15-2-4-22-3-1-14-11(21)8-5-9(20-19-8)17-10(7)18-12/h5-6H,1-4H2,(H,14,21)(H3,15,16,17,18,19,20)
InChIKey: SXVMAWWNAHHYKH-UHFFFAOYSA-N
Selectivity profile of JA310 was determined with the selectivity profiling assay against 340 wild-type protein kinases from Reaction Biology at 1000 nM and on- and off targets were evaluated in cellulo EC50 with NanoBRET assay.
Kinase | Percent of control(%) @ 1000 nM | NanoBRET EC50 (µM) |
| MST3 | 13.5 | 0.11 |
| MST4 | 18.0 | 1.43 |
| LIMK1 | 36.6 | 5.67 |
| LIMK2 | 38.2 | 1.78 |
| PKCt | 42.6 | > 50.0 |
| MERTK | 44.2 | 5.14 |
| MAP3K11 | 45.8 | > 50.0 |
| MELK | 47.0 | 45.7 |
| STK39 | 49.5 | n.d. |
The negative control JA262 showed no activity on a DSF assay for 102 kinases and no on-target activity determined by NanoBRET in intact cells and in lysed cells.
ITC on MST4: KD = 116 nM
JA310 (21c) is metabolic stable. JA310 (21c) shows no general cytotoxicity after 24h, but after 72h a decrease of the normalized cell count has been detected. At 1 µM JA310 (21c) shows slightly increase of cells in G1 Phase, whereas at higher concentration a S-Phase arrest has been observed.
[1] Thompson BJ, Sahai E. MST kinases in development and disease. J Cell Biol. 2015 Sep 14;210(6):871-82. doi: 10.1083/jcb.201507005.
[2] Cornils H, Kohler RS, Hergovich A, Hemmings BA. Human NDR kinases control G(1)/S cell cycle transition by directly regulating p21 stability. Mol Cell Biol. 2011 Apr;31(7):1382-95. doi: 10.1128/MCB.01216-10.
[3] Huang T, Kim CK, Alvarez AA, Pangeni RP, Wan X, Song X, Shi T, Yang Y, Sastry N, Horbinski CM, Lu S, Stupp R, Kessler JA, Nishikawa R, Nakano I, Sulman EP, Lu X, James CD, Yin XM, Hu B, Cheng SY. MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma. Cancer Cell. 2017 Dec 11;32(6):840-855.e8. doi: 10.1016/j.ccell.2017.11.005.
[4] Jennifer A. Amrhein, Lena M. Berger, Dimitrios-Ilias Balourdas, Andreas C. Joerger, Amelie Menge, Andreas Krämer, Julia M. Frischkorn, Benedict-Tilman Berger, Lewis Elson, Astrid Kaiser, Manfred Schubert-Zsilavecz, Susanne Müller, Stefan Knapp, Thomas Hanke. Synthesis of pyrazole-based macrocycles leads to a highly selective inhibitor for MST3. bioRxiv 2023.10.20.563248; doi: https://doi.org/10.1101/2023.10.20.563248
Binding mode of JA310 in complex with MST3 was determined (PDB 8QLQ). The inhibitor binds in an orthosteric binding mode to the ATP pocket as a type I kinase inhibitor.
Government General Mary Simon has announced 53 new Order of Canada appointments- one of Canada’s highest honours. This prestigious recognition celebrates individuals from diverse backgrounds for their outstanding contributions to Canada.
Established in 1067, the Order of Canada acknowledges remarkable achievements, dedication to the community and service to the nation, with over 7,600 people spanning various sectors invested in making a difference in Canada.
TORONTO, Canada, October 3, 2023 – The Structural Genomics Consortium (SGC) today announced that it has entered into a partnership with Shanghai Stock Exchange listed company HitGen Inc. ("HitGen", SSE: 688222.SH). HitGen will utilize its DNA-encoded library (DEL) technology platform, specifically OpenDEL™, to screen under-represented targets chosen 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.
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.
| 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.
| 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 -20oC |
| 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 -20oC |
| 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
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.
Biological activity summary:
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.
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.