Synthesising Positive and Negative Controls

[mattodd]

[daniellehanke]

Brief background surrounding reported ABHD6 inhibitors (two main classes: carbamates and triazole urea derivatives)

Most promising positive control compounds:
Triazole ureas 9 (KT182), 11 (KT185), and 20 (KT203) from J. Med. Chem., 2013, 56(21), 8270-8279, https://doi.org/10.1021/jm400899c

• Each have sub-nanomolar IC50's in activity-based protein profiling (ABPP); most potent ABHD6 inhibitors reported to date

Some options for negative controls:
Triazole ureas KT116 and/or KT117 from Nat. Chem. Biol. 2012, 8, 999–1007, https://doi.org/10.1038/nchembio.1105 (demonstrated weakest ABHD6 inhibition)

• Both are intermediates formed in synthesis of 20 which would simplify synthesis
• SI from paper show results from competitive ABPP with ABHD6, but no IC50 derived; from gels, looks like KT117 is weaker (minimal inhibition below 80 nM)

Triazole ureas DH376 and/or DO34 from PNAS 2016, 113(1), 26-33, https://doi.org/10.1073/pnas.1522364112 (DAGLa/b inhibitors with weak ABHD6 inhibition)

• Similar/possibly weaker inhibition of ABHD6 than KT116 or KT117 (IC50 values 125-250 nM in competitive ABPP experiments; calculated in the SI)
• Similar synthetic workflow to positive control compounds above (DO34 easier synthesis of the two)

Carbamates from J. Am. Chem. Soc. 2007, 129(31), 9594–9595, https://doi.org/10.1021/ja073650c that did not show up as hits in the initial screen

• Of the ~50 carbamate compounds were initially screened in ABPP for ABHD6 at 50 uM, only three showed up as hits (5, 16, and 18); remaining compounds could be explored to use as negative controls
• Different scaffold from positive control compounds so maybe not ideal, but seem to be much weaker inhibitors which is favourable (no inhibition up to 50 uM)
• Couple examples:

Brief lit review summarized in following document (papers referenced above highlighted yellow):

Reported ABHD6 Inhibitors_Summary Oct 2025.docx

[daniellehanke]

Another idea about the positive and negative control compounds: a 2017 MedChemComm paper (https://doi.org/10.1039/C7MD00029D) explored the SAR of previously reported dual DAGL/ABHD6 inhibitors, focusing on the chirality at the C2 position.

The R-enantiomer of KT109 shows potent ABHD6 inhibition (IC50 2.5nM), whereas the S-enantiomer does not (IC50 630 nM).

Both enantiomers also inhibit DAGLalpha (IC50's for R and S enantiomers are 0.8 nM and 40 nM, respectively); but if we are using these control compounds in biochemical screens with purified ABHD6, the selectivity shouldn't be an issue.