DO264

Protein target name:

ABHD12

Mechanism of action:

Reversible Competitive Inhibitor

Recommended Concentration for use in cells:

~~up to 1 uM~~

Reviewer suggested Concentration for use in cells:

20-200 nM is ideal, up to 1 uM acceptable

Probe Availability

MedChemExpress

Probe Information

Target and Probe Information

Target Details

Target class:

Lipid metabolism

Subclass:

Serine Esterase

HUGO ID:

HGNC:15868

Entrez gene ID:

26090

UNIPROT ID:

Q8N2K0

Target alias:

PHARC; ABHD12A; BEM46L2; hABHD12; C20orf22; dJ965G21.2

Probe Details

Chemical Probes Portal ID:

CPP1851

Probe alias:

Compound 46, DO-264

PubChem CID:

134813646

SMILES string:

S=C(NC1=CC=CN=C1)NC2CCN(C3=NC=CC(OC4=CC=C(OC(F)(F)F)C=C4Cl)=C3Cl)CC2

InChi Key:

PIEQDBPBBQPLPL-UHFFFAOYSA-N

Control compound:

DO271

Physico-chemical properties

NMR:

1H NMR (CDCl3, 600 MHz) δ 8.56 – 8.55 (m, 2H), 7.98 (d, J = 5.6 Hz, 1H), 7.84 (s, 1H), 7.64 (d, J = 8.3 Hz, 0H), 7.41 – 7.38 (m, 2H), 7.18 (ddd, J = 9.0, 2.8, 0.9 Hz, 1H), 7.12 (d, J = 8.9 Hz, 1H), 6.17 (d, J = 5.6 Hz, 1H), 5.87 (d, J = 7.9 Hz, 1H), 4.55 (s, 1H), 3.82 (d, J = 12.9 Hz, 2H), 3.05 (ddd, J = 13.5, 11.4, 2.5 Hz, 2H), 2.26 – 2.22 (m, 2H), 1.66 – 1.60 (m, 2H).

13C NMR (CDCl3, 150 MHz) 179.54, 160.07, 159.89, 148.32, 147.28, 145.78, 145.72, 145.71, 133.19, 132.07, 127.18, 123.96, 123.44, 122.48, 120.50, 119.83 (q, J = 257.1 Hz, OCF3), 110.66, 105.25, 52.00, 47.77, 31.27.

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

IC50

11 nM

Potency assay:

Inhibition of lyso-PS hydrolysis

Structure-activity relationship:

yes

Off target activity

Probe Selectivity in Vitro:

ABPP studies of DO264 (46) and other representative (thio)urea inhibitors of ABHD12 confirmed excellent selectivity over other serine hydrolases including phosphatidylserine lipases, such PS-PLA1o and ABHD16A and the PLA1-type PI lipase DDHD1.

In cell validation

On target activity in cells

Potency in cells:

IC50

26 nM

Potency assay (cells):

Gel-Based ABPP

Target engagement assay (cells):

Quantification by targeted LC-MS analysis of the lyso-PS

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

Assessment of cytotoxicity in human cell lines (THP1, HT1080, MDA-MB-231, T98G) treated with DO264 across a concentration of 0.1-10 μM and cell viability measured 24 h later using CellTighter-Glo®.

In vivo validation

Organism:

C57BL/6 mice

Dose:

30 mg/Kg60 mg/Kg

Route of delivery:

OralIntraperitoneal

Organ of Interest

Organ (O):

Brain

Primary Reference

Reference (doi):

10.1038/s41589-018-0155-8 10.1021/acs.jmedchem.8b01958 10.1021/acschembio.0c00086

Reference (PMID):

30420694 30720278 32195565

Under Review

This probe is in the process of SAB review. Please continue to check back for new reviews and commentary.

SAB Rating

Rating for use in Cells

2 review(s)

Rating for use in Model Organisms

2 review(s)

SAB Members

SAB Comments

DO264 is a high quality probe for reversible inhibition of human and mouse lyso-PS lipase ABHD12. The probe is effective and has excellent selectivity in cell lysates, in cell culture models, and can be used in in vivo experiments to block ABHD12 activity in the mouse brain. Gel-based competitive ABPP analysis indicates that DO264 also engages with ABHD12 in liver, spleen and lung tissues. Use of an activity-based probe with quantitative mass spectrometry and competitive in-gel analyses demonstrate excellent selectivity among endogenous serine hydrolases when used at the recommended concentrations. An inactive control analog (S)-DO271 provides a selectivity control.

Aug 13 2020 - 1:17pm

This probe is of reasonable high quality, but there are two concerns, based on the lack of available data (as opposed to existing negative data). 1) How selective is the probe outside of the serine hydrolase family? The data reported is using the FP probe, which only allows in-family comparisons. 2) What is the PK of the compound, when dosed IP and PO? With unknown broad selectivity and unknown exposure, there is some concern that in vivo data will be misinterpreted.

Sep 5 2020 - 8:30am

DO264

Probe Availability

Probe Information

Target Details

PHARC; ABHD12A; BEM46L2; hABHD12; C20orf22; dJ965G21.2

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

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DO264

Probe Availability

Probe Information

Target Details

PHARC; ABHD12A; BEM46L2; hABHD12; C20orf22; dJ965G21.2

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?