A-196

Reagent Authentication Certificate:

Chemical Probes Portal A-196.pdf

Protein target name:

KMT5B, KMT5C

Mechanism of action:

Substrate-competitive inhibitorSAM non-competitive inhibitor

Recommended Concentration for use in cells:

500-1000 nM

Probe Availability

MedChem Express (A-196)

Probe Information

Target and Probe Information

Target Details

Target class:

Epigenetics

Subclass:

Protein methyltransferase

HUGO ID:

HGNC:24283 HGNC:28405

Entrez gene ID:

51111 84787

UNIPROT ID:

Q4FZB7 Q86Y97

Target alias:

Lysine methyltransferase 5B, CGI-85, SUV420H1, SUV4-20H1, lysine methyltransferase 5C, SUV420H2, Suv4-20h2

Probe Details

Chemical Probes Portal ID:

CPP58

PubChem CID:

117072548

SMILES string:

ClC1=C(Cl)C=C(C(C2=CC=NC=C2)=NN=C3NC4CCCC4)C3=C1

InChi Key:

ABGOSOMRWSYAOB-UHFFFAOYSA-N

Control compound:

SGC2043, A-197

Physico-chemical properties

Solubility - preferred solvent:

DMSO

Solubility - concentration:

20 mM

NMR:

1H NMR (CDCl3, 400 MHz) δ 8.81-8.79 (dd, J1 = 1.6 Hz, J2 = 4.4 Hz, 2H), 8.01 (s, 1H), 7.94 (s, 1H), 7.63-7.61 (dd, J1 = 1.6 Hz, J2 = 4.4 Hz, 2H), 5.15 (d, J = 6.4 Hz, 1H), 4.79-4.71 (m, 1H), 2.35-2.27 (m, 2H), 1.84-1.66 (m, 4H), 1.64-1.59 (m, 2H);

13C NMR (100 MHz, DMSO-d6) δ 152.5, 150.3 (2C), 147.6, 144.2, 135.2, 134.4, 126.8, 125.6, 124.9, 124.6 (2C), 117.5, 53.2, 32.5 (2C), 24.2 (2C).

Storage Recommendations

Storage (dry powder):

-20 oC for 2 years

Storage (solution):

-80 oC (5-10 uL aliquots) for 6 months

Storage sensitivities:

Limit to one freeze-thaw cycle

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

IC50

25 nM (SUV420H1), 144 nM (SUV420H2)

Potency assay:

Scintillation proximity assay - Methyltransferase activity assays for SUV420H1 and SUV420H2 were performed by monitoring the incorporation of tritium-labeled methyl group to monomethylated lysine 20 of peptide H4(1-24) (H4K20Me1). The experiments were performed in triplicate.

PDB ID for probe-target interaction (3D structure):

5CPR

Structure-activity relationship:

Yes, to be published separately

Off target activity

Potency (off target):

IC50

Potency assay (off target):

Scintillation proximity assay. No activity was detected in assays with 29 methyltransferases at 1 or 10 uM. No activity was detected against other epigenetic proteins (chromatin binders and readers, including 53BP1 and WDR5).

Probe Selectivity in Vitro:

125 non-family targets including kinases, GPCRs, ion channels, and transporters (via Cerep and the PDSP panels) were screened. Significant inhibition was observed at 10 uM for six of these targets: Adenosine receptors A1 (Ki=0.021 uM), A2 (Ki=0.028 uM), A3 (Ki=2 uM), GABA-gated chloride channel (Ki=1.8 uM), NK2 (Ki=4.1 uM), DOP (Ki=9 uM).

In cell validation

On target activity in cells

Potency in cells:

EC50

740 nM (activating H4K20me1), 262 nM (inhibiting H4K20me2), 370 nM (inhibiting H4K20me3)

Potency assay (cells):

In U2OS cells, EC50s were determined by western blot after treating the cells for 48 hours. Comparable results were obtained in LnCaP cells. The cellular effects were also evaluated using high-content microscopy, which allowed the simultaneous assessment of two methyl marks. In the assay (in U2OS cells), IC50 for H4K20me1=1.91 uM (activation), H4K20me3=0.502 uM (inhibition). Comparable data were obtained in PC3 cells.

Target engagement assay (cells):

Indirect, impact on substrate methylation

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

A-196 was not toxic up to 12.5 uM in PC-3, U2OS and HeLa cells.

Primary Reference

Reference (doi):

10.1038/nchembio.2282

Reference (PMID):

28114273

Endorsed probe

The Chemical Probes Portal endorses this compound as chemical probe for use in cells.

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

3 review(s)

SAB Members

SAB Comments

This probe represents the best in class for SUV420H1 and H2 - hitting the enzymes at 25 and 144 nM, respectively, with selectivity data against a wide panel of related epigenetic and other targets. The originating paper gives good confidence around this molecule and includes strong biochemical, biophysical, structural & cellular data, as well as structurally related negative controls. No data are presented for the wider pharmacokinetic, drug-like profiles, and so no conclusion can be drawn on its suitability as a probe for use in model organism studies. However, permeability from cellular studies and chemical structure would support its further investigation towards these studies.

Mar 30 2017 - 6:50pm

A-196

Probe Availability

Probe Information

Target Details

Lysine methyltransferase 5B, CGI-85, SUV420H1, SUV4-20H1, lysine methyltransferase 5C, SUV420H2, Suv4-20h2

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

Primary Reference

Supporting Organizations

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A-196

Probe Availability

Probe Information

Target Details

Lysine methyltransferase 5B, CGI-85, SUV420H1, SUV4-20H1, lysine methyltransferase 5C, SUV420H2, Suv4-20h2

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

Primary Reference

Supporting Organizations

Interested in supporting the portal?