18.07.2023

Support for the Characterization and Validation of Renewable Antibodies

by: SGC

At Structural Genomics Consortium, we are committed to driving advancements in basic and biomedical research that can positively impact the world. We believe that collaborative efforts and innovative solutions are essential for achieving groundbreaking discoveries. Today, we invite researchers from all corners of the globe to stand united in support of renewable antibody resources.

Sign the Petition to Prioritize Renewable Antibody Resources!

Have you ever experienced any of the following research roadblocks?

TH-257 A chemical probe for LIMK1/2

This probe is available from Sigma and Cayman Chemical.

Its negative control (TH-263) is available for purchase from Cayman Chemical.

overview
properties
references

overview

Probe		Negative control

TH-257		TH-263

LIM kinases belong to the family of cytoplasmic tyrosine-like kinases with dual specificity (serine/threonine and tyrosine). However, known LIMK substrate are usually phosphorylated at serine and threonine residues LIM kinases comprises LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) which show 50% sequence identity in human. Both LIMK1 and LIMK2 present with a unique domain organization containing two N-terminal LIM domains, a PDZ domain, a proline/serine-rich domain and a C-terminal kinase domain [1].

Both proteins are expressed widely in embryonic and adult tissues, but show some cell-type specific expression. Accordingly, the two kinases have overlapping functions, but appear non-redundant. Knockout studies in mice show that LIMK1 is required for development of the central nervous system [2], whereas LIMK2 knockout impairs the activity of testicular germ cells [3].

LIMKs are effectors of cell morphology and motility and apoptosis by regulating the actin cytoskeleton. The LIMKs signal downstream from Rho GTPases and are activated by phosphorylation of the activation loop by upstream kinases, including Rho kinase (ROCK), PAK1/2/4 and MRCKα. The best characterized LIMK substrates are cofilin1 (non-muscle cofilin), cofilin2 (muscle cofilin) and destrin (actin depolymerizing factor, ADF). Phosphorylation of cofilin serine-3 inactivates the actin severing ability promoting F-actin polymerization, stress fibre formation and focal adhesion formation [4].

LIM kinases can shuttle between the cytoplasm and the nuclear compartment of a cell, a process tightly regulated by association with other partners such as p57^kip2 and phosphorylation in the activation segment by PAK kinases [1]. Inhibition of LIMK hyper-stabilizes mitotic spindles inducing a G2/M cell cycle block suggesting an important role for these kinases in microtubule dynamics [5].

Increased phosphorylation of LIMK1 has been reported in neurons in areas affected with Alzheimer Disease [6]. LIM kinases play important roles in cancer metastasis like highly invasive prostate and breast cancer, which is reversed by gene silencing [7, 8]. LIMK1 overexpression is also found in malignant melanoma, as well as most tumour cell lines. Other applications for LIMK inhibitors are open-angle glaucoma [9]. In addition, LIMK1 interacts with the long isoform of the type II bonemorphogenetic protein (BMP) receptor contributing to the pathology of Fragile X syndrome, a common inherited form of intellectual disability [10].

TH-257 is a chemical probe for LIMK1 and LIMK2. TH-257 is an allosteric inhibitor targeting a binding pocket induced by an αC and DFG-out conformation. It potently inhibits cofilin phosphorylation with an IC50 of 84 nM for LIMK1 and 39 nM for LIMK2 in a RapidFire MS assay. TH257 is exquisitely selective and no significant activity against the wider kinome has been observed in the KINOMEscan assay (Dx) at 1 μM (IC50 >> 50 % inhibition). In a life cell NanoBRET assay (Promega) TH257 has an IC50 of 250 nM against ectopically expressed full-length LIMK1 and 150 nM LIMK2, respectively.

A chemically related negative control compound, TH-263, is provided.

properties

Probe		Negative control

TH-257		TH-263

Physical and chemical properties for TH-257
Molecular weight	422.2
Molecular formula	C24H26N2O3S
MollogP	5.138
PSA	57.08
No. of chiral centres	0
No. of rotatable bonds	10
No. of hydrogen bond acceptors	6
No. of hydrogen bond donors	1

Physical and chemical properties for TH-263 (Negative Control)
Molecular weight	380.1
Molecular formula	C21H20N2O3S
MollogP	3.636
PSA	66.41
No. of chiral centres	0
No. of rotatable bonds	8
No. of hydrogen bond acceptors	7
No. of hydrogen bond donors	2

SMILES:
TH-257: CCCCN(C(C1=CC=C(S(NC2=CC=CC=C2)(=O)=O)C=C1)=O)CC3=CC=CC=C3
TH-263: O=S(C1=CC=C(C=C1)C(NCC2=CC=CC=C2)=O)(NCC3=CC=CC=C3)=O

InChI:
TH-257: InChI=1S/C24H26N2O3S/c1-2-3-18-26(19-20-10-6-4-7-11-20)24(27)21-14-16-23(17-15-21)30(28,29)25-22-12-8-5-9-13-22/h4-17,25H,2-3,18-19H2,1H3

TH-263: InChI=1S/C21H20N2O3S/c24-21(22-15-17-7-3-1-4-8-17)19-11-13-20(14-12-19)27(25,26)23-16-18-9-5-2-6-10-18/h1-14,23H,15-16H2,(H,22,24)

InChIKey:
TH-257: VNCIWNGCMAKKEO-UHFFFAOYSA-N
TH-263: QDGVJMITKNOVTP-UHFFFAOYSA-N

selectivity profile

in vitro potency

cell based assay data

references

Manetti F. LIM kinases are attractive targets with many macromolecular partners and only a few small molecule regulators. Med Res Rev 2012;32(5):968-998.
Meng Y, Zhang Y, Tregoubov V, Janus C, Cruz L, Jackson M, Lu WY, MacDonald JF, Wang JY, Falls DL et al. Abnormal spine morphology and enhanced LTP in LIMK-1 knockout mice. Neuron 2002;35(1):121-133.
Takahashi H, Koshimizu U, Miyazaki J, Nakamura T. Impaired spermatogenic ability of testicular germ cells in mice deficient in the LIM-kinase 2 gene. Dev Biol 2002;241(2):259-272.
Bernard O. Lim kinases, regulators of actin dynamics. Int J Biochem Cell Biol 2007;39(6):1071-1076.
Oku Y, Tareyanagi C, Takaya S, Osaka S, Ujiie H, Yoshida K, Nishiya N, Uehara Y. Multimodal effects of small molecule ROCK and LIMK inhibitors on mitosis, and their implication as anti-leukemia agents. PLoS One 2014;9(3):e92402.
Heredia L, Helguera P, de Olmos S, Kedikian G, Sola Vigo F, LaFerla F, Staufenbiel M, de Olmos J, Busciglio J, Caceres A et al. Phosphorylation of actin-depolymerizing factor/cofilin by LIM-kinase mediates amyloid beta-induced degeneration: a potential mechanism of neuronal dystrophy in Alzheimer's disease. J Neurosci 2006;26(24):6533-6542.
Yoshioka K, Foletta V, Bernard O, Itoh K. A role for LIM kinase in cancer invasion. Proc Natl Acad Sci U S A 2003;100(12):7247-7252.
Davila M, Frost AR, Grizzle WE, Chakrabarti R. LIM kinase 1 is essential for the invasive growth of prostate epithelial cells: implications in prostate cancer. J Biol Chem 2003;278(38):36868-36875.
Harrison BA, Almstead ZY, Burgoon H, Gardyan M, Goodwin NC, Healy J, Liu Y, Mabon R, Marinelli B, Samala L et al. Discovery and Development of LX7101, a Dual LIM-Kinase and ROCK Inhibitor for the Treatment of Glaucoma. ACS Med Chem Lett 2015;6(1):84-88.
Kashima R, Roy S, Ascano M, Martinez-Cerdeno V, Ariza-Torres J, Kim S, Louie J, Lu Y, Leyton P, Bloch KD et al. Augmented noncanonical BMP type II receptor signaling mediates the synaptic abnormality of fragile X syndrome. Sci Signal 2016;9(431):ra58.

pk properties

co-crystal structures

synthetic schemes

materials and methods

JA397 A chemical probe for the TAIRE family

overview
properties
selectivity profile
cell based assay data
references

overview

Probe		Negative control

JA397		JA314

The TAIRE family of protein kinases are the cyclin-dependent kinases (CDK14-18), which are Ser/Thr kinases and belong to the CMGC family. The CDKs are activated by specific cyclins and are known to play an important role in cell cycle regulation (Wood 2018). Therefore, CDKs are now an important protein family that also has therapeutic significance, as demonstrated by the recent FDA-approved drugs for CDK4/6. However, for the TAIRE family little is known. However, the TAIRE family is less well described in the literature. It can be further subdivided into the PFTAIRE family (CDK14-15) and PCTAIRE family (CDK16-18). There is various evidence, for example, that CDK14 is involved in the WNT signalling pathway (Davidson, 2010); CDK15 regulates the beta-catenin/MEK-ERK signalling pathway (Huang, 2015), among others; CDK16 has an effect on cell cycle through phosphorylation of P27 (Yanagi, 2016); CDK17 plays a role in glycerophospholipid metabolism (Liu, 2017); and CDK18 regulates cell motility through the FAK/RhoA/ROCK signalling pathway (Matsuda, 2017).

The SGC has developed JA397, a potent and selective inhibitor for the TAIRE family with cellular activity, ranging from IC50 values of 21 _nM to 307 _nM as determined by NanoBRET.

The chemical probe (JA397) is accompanied by a negative control (JA314) that is structurally closely related to the probe molecule.

Potency Against Target Family

JA397 had an EC₅₀ of 27.1 _nM, 252 _nM, 39.0 _nM, 77.2 _nM and 172 _nM to CDK14, CDK15, CDK16, CDK17 and CDK18, respectively in the NanoBRET-lysed mode assay.

Selectivity

JA397 was selective in an in vitro kinase panel from Reaction Biology at 1 µM against 340 WT Kinases, followed by cellular NanoBRET assays. Selectivity within the CDK family was determined by NanoBRET.

Dosage

Based on the potency and the selectivity of the chemical probe and to minimize the risk of unspecific cytotoxicity, we recommend a concentration of no higher than 1 µM for cell-based assays.

Cellular Activity

JA397 displayed an EC₅₀ of 72.1 _nM, 307 _nM, 33.4 _nM, 21.2 _nM and 121 _nM on CDK14, CDK15, CDK16, CDK 17 and CDK18 respectively in intact cells in the NanoBRET assay.

properties

Probe		Negative control

JA397		JA314

Physical and chemical properties JA397
Molecular weight	481.56
Molecular formula	C₂₄H₃₁N₇O₄
IUPAC name	tert-butyl 3-((2-((4-((tert-butoxycarbonyl)amino)benzyl)amino)pyrimidin-4-yl)amino)-1H-pyrazole-5-carboxylate
clogP	4.86
tPSA	137.8
No. of chiral centres	0
No. of rotatable bonds	11
No. of hydrogen bond acceptors	10
No. of hydrogen bond donors	4
Storage	r. t.

SMILES: O=C(C1=CC(NC2=NC(NCC3=CC=C(C=C3)NC(OC(C)(C)C)=O)=NC=C2)=NN1)OC(C)(C)C

InChI: InChI=1S/C24H31N7O4/c1-23(2,3)34-20(32)17-13-19(31-30-17)28-18-11-12-25-21(29-18)26-14-15-7-9-16(10-8-15)27-22(33)35-24(4,5)6/h7-13H,14H2,1-6H3,(H,27,33)(H3,25,26,28,29,30,31)

InChIKey: JQLMEZBHIJSVKR-UHFFFAOYSA-N

Physical and chemical properties JA314
Molecular weight	452,52
Molecular formula	C₂₂H₂₈N₈O₃
IUPAC name	tert-butyl (4-((4-((5-(methylcarbamoyl)-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)amino)phenethyl)carbamate
clogP	3.55
tPSA	140.6
No. of chiral centres	0
No. of rotatable bonds	10
No. of hydrogen bond acceptors	9
No. of hydrogen bond donors	5
Storage	r. t.

SMILES: O=C(C1=CC(NC2=NC(NC3=CC=C(C=C3)CCNC(OC(C)(C)C)=O)=NC=C2)=NN1)NC

InChI: InChI=1S/C22H28N8O3/c1-22(2,3)33-21(32)25-11-9-14-5-7-15(8-6-14)26-20-24-12-10-17(28-20)27-18-13-16(29-30-18)19(31)23-4/h5-8,10,12-13H,9,11H2,1-4H3,(H,23,31)(H,25,32)(H3,24,26,27,28,29,30)

InChIKey: VJWCJKXRULCPCJ-UHFFFAOYSA-N

selectivity profile

Kinome-wide selectivity profile of JA397 was determined in our in-house kinase DSF-panel comprising 105 kinases and at Reaction Biology at 1 µM comprising 340 WT kinases.

The selectivity against a CDK family was determined by NanoBRET.

DSF-Panel against 105 kinases:

Reaction Biology (340 WT kinases) @ 1µM:

Selectivity within the CDK-Family:

The negative control JA314 showed no stabilization against 105 kinases screened in our in-house DSF-Panel.

DSF-Panel against 105 kinases:

in vitro potency

cell based assay data

JA397 displayed an EC₅₀ values ranging from 21 _nM to 307 _nM against the TAIRE family, determined by NanoBRET^TM assay.

The negative control compound 314 displayed a EC₅₀ value of 4165 _nM against CDK16, determined by NanoBRET^TM assay.

references

Amrhein JA, Berger LM, Tjaden A, Krämer A, Elson L, Tolvanen T, Martinez-Molina D, Kaiser A, Schubert-Zsilavecz M, Müller S, Knapp S, Hanke T. Discovery of 3-Amino-1H-pyrazole-Based Kinase Inhibitors to Illuminate the Understudied PCTAIRE Family. Int J Mol Sci. 2022 Nov 27;23(23):14834. doi: 10.3390/ijms232314834. PMID: 36499165; PMCID: PMC9736855.

Davidson G, Niehrs C. Emerging links between CDK cell cycle regulators and Wnt signaling. Trends Cell Biol. 2010 Aug;20(8):453-60. doi: 10.1016/j.tcb.2010.05.002. Epub 2010 Jun 4. PMID: 20627573.

Huang C, Du R, Jia X, Liu K, Qiao Y, Wu Q, Yao N, Yang L, Zhou L, Liu X, Xiang P, Xin M, Wang Y, Chen X, Kim DJ, Dong Z, Li X. CDK15 promotes colorectal cancer progression via phosphorylating PAK4 and regulating β-catenin/ MEK-ERK signaling pathway. Cell Death Differ. 2022 Jan;29(1):14-27. doi: 10.1038/s41418-021-00828-6. Epub 2021 Jul 14. PMID: 34262144; PMCID: PMC8738751.

Liu M, Xu Z, Du Z, Wu B, Jin T, Xu K, Xu L, Li E, Xu H. The Identification of Key Genes and Pathways in Glioma by Bioinformatics Analysis. J Immunol Res. 2017;2017:1278081. doi: 10.1155/2017/1278081. Epub 2017 Dec 6. PMID: 29362722; PMCID: PMC5736927.

Matsuda S, Kawamoto K, Miyamoto K, Tsuji A, Yuasa K. PCTK3/CDK18 regulates cell migration and adhesion by negatively modulating FAK activity. Sci Rep. 2017 Mar 31;7:45545. doi: 10.1038/srep45545. PMID: 28361970; PMCID: PMC5374530.

Wood DJ, Endicott JA. Structural insights into the functional diversity of the CDK-cyclin family. Open Biol. 2018 Sep;8(9):180112. doi: 10.1098/rsob.180112. PMID: 30185601; PMCID: PMC6170502.

Yanagi T, Matsuzawa S. PCTAIRE1/PCTK1/CDK16: a new oncotarget? Cell Cycle. 2015;14(4):463-4. doi: 10.1080/15384101.2015.1006539. PMID: 25590439; PMCID: PMC4347670.

pk properties

co-crystal structures

synthetic schemes

materials and methods

26.06.2023

June as the Alzheimer’s Disease and Brain Awareness Month: Spotlight on the Brilliant Minds behind SGC’s Brain Research

by: SGC

Alzheimer’s Disease and brain awareness month in June provides a significant opportunity to recognize the critical importance of brain research and its profound impact on global health. Through an open science model, SGC is dedicated to unravelling the complexities of neurological disorders that affect over 1 billion people worldwide – diseases that include Alzheimer's and Parkinson's disease, amyotrophic lateral sclerosis (ALS), and rare neurodegenerative diseases like Huntington’s disease.

09.06.2023

Celebrating 20 years of SGC

by: SGC

Let's continue to collaborate and push the boundaries to open science together!

Tweet your story or post it on LinkedIn using: #20yearsSGC

31.05.2023

SGC’s Donated Chemical Probes Program: Driving Drug Discovery and Target Biology

by: SGC

In the pursuit of understanding the function of all human proteins, it is essential to employ various tools, including the use of potent, selective, and broadly characterized small molecule modulators, known as chemical probes. These chemical probes are often described as one of the most versatile tools to explore the role of a specific protein in complex biological systems and advance existing knowledge about the protein and its relevance for therapeutic development. In this dynamic realm of drug discovery, breakthroughs often stem from collaborative efforts and innovative resources.

24.05.2023

Join our global network with a Mitacs program

by: SGC

Trainees are an integral part of the 20 years SGC global network of over 250 scientists. Since 2015, we've proudly partnered with Mitacs, offering over 550 internship units to trainees across 25+ unique projects, totaling $8.4M. This direct support funds trainee stipends and research costs.

10.05.2023

Structural Genomics Consortium at UNC receives $1.5 million grant to help find possible new treatment options for patients with ALS.

by: SGC

North Carolina, May 10, 2023 – The Structural Genomics Consortium (SGC) at the University of North Carolina at Chapel Hill has been awarded a $1.5 million grant from the U.S Department of Defense to support drug discovery efforts aimed at the investigation of small molecules that could potentially serve as new treatment options for patients with Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s Disease.

SGC-CK2-2 A chemical probe for CK2/CSNK2.

The probe is available from Sigma.

The control can be requested by clicking here.

overview
properties
selectivity profile
cell based assay data
references

overview

Probe		Negative control

SGC-CK2-2		SGK-CK2-2N

From a library of naphthyridines, we identified a potent and cell-active chemical probe (SGC-CK2-2) that inhibits casein kinase 2 (CK2/CSNK2). Comprehensive evaluation of kinome-wide selectivity confirmed that this CK2 probe demonstrates excellent selectivity. A structurally similar naphthyridine (SGC-CK2-2N) was characterized as a negative control that does not inhibit CK2 and exhibits exceptional selectivity when profiled broadly. Our CK2 chemical probe is not broadly antiproliferative when tested against 16 cancer cell lines of diverse origins. Versus a published CK2 chemical probe (SGC-CK2-1), our scaffold is a distinct chemotype, has non-overlapping kinase off-targets, and demonstrates improved kinetic solubility. Our chemical probe set can be used by the community to further characterize the diverse roles of CK2.

Biological activity summary:

Enzymatic assay (Eurofins): CK2⍺ IC₅₀ = 3.0 nM; CK2⍺ IC₅₀ <1.0 nM
Cellular data (NanoBRET): CK2⍺ IC₅₀ = 920 nM; CK2⍺’ IC₅₀ = 200 nM
Only 3/403 kinases with PoC <10 when screened at 1 μM

properties

Probe		Negative control

SGC-CK2-2		SGK-CK2-2N

Physical and chemical properties for SGC-CK2-2
Molecular weight	330.35
Molecular formula	C₁₉H₁₄N₄O₂
IUPAC name	5-(benzylamino)pyrimido[4,5-c]quinoline-8-carboxylic acid
MollogP	3.11
PSA	86.41
No. of chiral centers	0
No. of rotatable bonds	4
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 100 mM) are stable at -20^oC
Dissolution	Soluble in DMSO up to 100 mM

Physical and chemical properties for SGC-CK2-2N
Molecular weight	329.36
Molecular formula	C₂₀H₁₅N₃O₂
IUPAC name	5-(benzylamino)benzo[f][1,7]naphthyridine-8-carboxylic acid
MollogP	3.7
PSA	74.05
No. of chiral centers	0
No. of rotatable bonds	4
No. of hydrogen bond acceptors	4
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

SMILES:

SGC-CK2-2: OC(C1=CC2=NC(NCC3=CC=CC=C3)=C4N=CN=CC4=C2C=C1)=O

SGC-CK2-2N: OC(C1=CC2=NC(NCC3=CC=CC=C3)=C4N=CC=CC4=C2C=C1)=O

InChI:

SGC-CK2-2: InChI=1S/C19H14N4O2/c24-19(25)13-6-7-14-15-10-20-11-22-17(15)18(23-16(14)8-13)21-9-12-4-2-1-3-5-12/h1-8,10-11H,9H2,(H,21,23)(H,24,25)

SGC-CK2-2N: InChI=1S/C20H15N3O2/c24-20(25)14-8-9-15-16-7-4-10-21-18(16)19(23-17(15)11-14)22-12-13-5-2-1-3-6-13/h1-11H,12H2,(H,22,23)(H,24,25)

InChIKey:

SGC-CK2-2: HEVVNKYNJCSHFA-UHFFFAOYSA-N

SGC-CK2-2N: VBKHXXPYOSWXSA-UHFFFAOYSA-N

selectivity profile

Selectivity Profile

SGC-CK2-2 was profiled in the DiscoverX scanMAX assay against 403 wild-type kinases at 1 μM. Only 3 kinases showed PoC <10 giving an S₁₀(1 μM) = 0.007. When the PoC <35 fraction was examined, 13 kinases were included (S₃₅(1 μM) = 0.032). Potential off-targets within the S₃₅(1 μM) fraction were tested via biochemical enzymatic assays plus NanoBRET target engagement assays for CK2⍺ and CK2⍺’. Data corresponding with off-target kinase activity is shown in the table below.

SGC-CK2-2N was also tested in the DiscoverX scanMAX panel and 1 kinase had a PoC <35 (S₃₅(1 μM) = 0.002). The negative control was sent to Eurofins for testing in the enzyme assays for PIP5K1C, CK2⍺, and CK2⍺’. All results are in the table below.

in vitro potency

cell based assay data

A NanoBRET assay was utilized to assess the binding affinity of SGC-CK2-2 to CK2⍺ and CK2⍺’. The negative control shows no binding affinity for CK2⍺ or CK2⍺’.

references

Davis-Gilbert, Z. W.; Krämer, A.; Dunford, J. E.; Howell, S.; Senbabaoglu, F.; Wells, C. I.; Bashore, F. M.; Havener, T. M.; Smith, J. L.; Hossain, M. A.; Oppermann, U.; Drewry, D. H.; Axtman, A. D. Discovery of a potent and selective naphthyridine-based chemical probe for casein kinase 2. ACS Med Chem Lett 2023, 14, 432–441; doi: 10.1021/acsmedchemlett.2c00530.

Davis-Gilbert, Z. W.; Krämer, A.; Dunford, J. E.; Howell, S.; Senbabaoglu, F.; Wells, C. I.; Havener, T. M.; Smith, J. L.; Hossain, M. A.; Oppermann, U.; Drewry, D. H.; Axtman, A. D. Discovery of a potent and selective naphthyridine-based chemical probe for casein kinase 2. ChemRxiv 2022, doi: 10.26434/chemrxiv-2022-05jcz.

pk properties

co-crystal structures

synthetic schemes

materials and methods

SGC-PI5P4Kγ/MYLK4-1 A chemical probe for PI5P4K gamma and MYLK4.

The probe and control can be requested by clicking here.

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

overview

Probe		Negative control

SGC-PI5P4Kγ/MYLK4-1		SGC-PI5P4Kγ/MYLK4-1N

From a library of indolyl pyrimidinamines, we identified a potent and cell-active chemical probe (SGC-PI5P4Kγ/MYLK4-1) that inhibits phosphatidylinositol-5-phosphate 4-kinase gamma (PI5P4Kγ) and myosin light chain kinase family member 4 (MYLK4). Comprehensive evaluation of kinome-wide selectivity confirmed that this chemical probe demonstrates excellent selectivity. A structurally similar indolyl pyrimidinamine (SGC-PI5P4Kγ/MYLK4-1N) was characterized as a negative control that does not inhibit PI5P4Kγ or MYLK4 and exhibits exceptional selectivity when profiled broadly. Our PI5P4Kγ/MYLK4 chemical probe increases mTORC1 signaling in MCF-7 cells without associated toxicity. Our chemical probe set can be used by the community to further explore the biology regulated by PI5P4Kγ and/or MYLK4.

properties

Probe		Negative control

SGC-PI5P4Kγ/MYLK4-1		SGC-PI5P4Kγ/MYLK4-1N

SMILES:

SGC-PI5P4Kγ/MYLK4-1: NC1=NC2=C(C=N1)CCCC3=C2C4=C(N3)C=CC(C5=CCOCC5)=C4

SGC-PI5P4Kγ/MYLK4-1N: NC1=NC2=C(C=N1)CCCCC3=C2C4=C(N3)C=CC(C#CC5CC5)=C4

InChI:

SGC-PI5P4Kγ/MYLK4-1: InChI=1S/C20H20N4O/c21-20-22-11-14-2-1-3-17-18(19(14)24-20)15-10-13(4-5-16(15)23-17)12-6-8-25-9-7-12/h4-6,10-11,23H,1-3,7-9H2,(H2,21,22,24)

SGC-PI5P4Kγ/MYLK4-1N: InChI=1S/C21H20N4/c22-21-23-12-15-3-1-2-4-18-19(20(15)25-21)16-11-14(8-7-13-5-6-13)9-10-17(16)24-18/h9-13,24H,1-6H2,(H2,22,23,25)

InChIKey:

SGC-PI5P4Kγ/MYLK4-1: VGFHPNRWHOQLEB-UHFFFAOYSA-N

SGC-PI5P4Kγ/MYLK4-1N: ZPQLOIBNJAOMDO-UHFFFAOYSA-N

selectivity profile

SGC-PI5P4Kγ/MYLK4-1 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) = 0.025). Potential off-targets within the S₃₅(1 μM) fraction were tested via biochemical enzymatic or binding assays and/or NanoBRET target engagement assays. Data corresponding with off-target kinase activity is shown in the table below.

Figure 2: SGC-PI5P4Kγ/MYLK4-1 was profiled in the DiscoverX scanMAX assay against 403 wild-type kinases at 1 μM and off-target kinases with PoC <35 were tested in an orthogonal assay. Rows colored green/yellow are PI5P4Kγ, MYLK4, PIKfyve, CLK2, and DYRK1A. No other kinases demonstrate enzymatic IC₅₀ values within 30-fold of the PI5P4Kγ K_d value. NB = NanoBRET

SGC-PI5P4Kγ/MYLK4-1N was also tested in the DiscoverX scanMAX panel and 5 kinases demonstrated PoC <35 (S₃₅(1 μM) = 0.012). The negative control was sent to SignalChem for testing in the enzyme assay for PIKfyve, to Eurofins DiscoverX or RBC for testing in the enzyme/binding assays for PI5P4Kγ (binding), RIPK5, MEK5, MEK4, and PAK2, and evaluated for MYLK4 affinity via NanoBRET assay. All results are in the table below.

Figure 3: SGC-PI5P4Kγ/MYLK4-1 was profiled in the DiscoverX scanMAX panel against 403 wild-type kinases at 1 μM and follow-up binding/enzymatic assays were done to confirm no activity. NB = NanoBRET

in vitro potency

Biological activity summary:

Enzymatic or binding assay (Eurofins DiscoverX): PI5P4Kγ K_d = 19 nM; MYLK4 IC₅₀ = 12 nM; PIKfyve IC₅₀ = 12 nM, CLK2 IC₅₀ = 370 nM; DYRK1A IC₅₀ = 520 nM

Figure 1: Kinome tree with PI5P4Kγ and MYLK4 highlighted as red circles. Illustration is reproduced courtesy of Eurofins DiscoverX (http://treespot.discoverx.com).

cell based assay data

A NanoBRET assay was utilized to assess the binding affinity of SGC-PI5P4Kγ/MYLK4-1 to PI5P4Kγ, MYLK4, PIKfyve, CLK2, DYRK1A, and TYK2 (JH2 domain). The negative control shows no/weak binding affinity for PI5P4Kγ and MYLK4.

Figure 4: SGC-PI5P4Kγ/MYLK4-1 was profiled in the PI5P4Kγ, MYLK4, PIKfyve, CLK2, DYRK1A, and TYK2 (JH2 domain) NanoBRET assays.

Figure 5: SGC-PI5P4Kγ/MYLK4-1N was profiled in the PI5P4Kγ, MYLK4, and PIKfyve NanoBRET assays.

references

Drewry, D. H.; Potjewyd, F. M.; Smith, J. L.; Howell, S.; Axtman, A. D. Identification of a chemical probe for lipid kinase phosphatidylinositol-5-phosphate 4-kinase gamma (PI5P4Kγ). Curr Res Chem Biol 2023, 3, 100036; doi: 10.1016/j.crchbi.2022.100036.

pk properties

co-crystal structures

synthetic schemes

materials and methods

Subscribe to