Probing the Interactions of Thiazole Abietane Inhibitors with the Human Serine Hydrolases ABHD16A and ABHD12

12-Thiazole abietanes are highly selective reversible inhibitors of hABHD16A that could potentially alleviate neuroinflammation. In this study, we used synthetic chemistry, competitive activity-based protein profiling, and computational methodologies to try to establish relevant structural determinants of activity and selectivity of this class of compounds for inhibiting ABHD16A over ABHD12. Five compounds significantly inhibited hABHD16A but also very efficiently discriminated between inhibition of hABHD16A and hABHD12, with compound 35 being the most effective, at 100 μM (55.1 ± 8.7%; p < 0.0001). However, an outstanding switch in the selectivity toward ABHD12 was observed in the presence of a ring A ester, if the C2′ position of the thiazole ring possessed a 1-hydroxyethyl group, as in compound 28. Although our data were inconclusive as to whether the observed enzyme inhibition is allosteric or not, we anticipate that the structure–activity relationships presented herein will inspire future drug discovery efforts in this field.

Membranes were isolated by passing the supernatant through a 25G needle 20 times, centrifuging at 100,000 × g for 1 h at 4 °C and resuspending the pellet in 500 μL ice-cold PBS.

Competitive activity-based protein profiling (cABPP) in HEK293T membrane proteomes
Total membrane proteomes were adjusted to 2 mg/mL in ice-cold PBS (pH 7.4) and incubated with compounds in DMSO at the indicated concentrations for 1 h followed by labelling with 2 µM azido-fluorophosphonate (ActivX™ Azido-FP Serine Hydrolase Probe, Thermo Scientific) for 1 h at room temperature in a total reaction volume of 50 μL.FP-labelled proteins were conjugated to an alkyne IR800 dye (LI-COR, Inc) by end-overend rotation in an equal volume of click chemistry reaction mix (5 μM IRDye 800CW alkyne infrared dye, 4 mM CuSO 4 , 400 μM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, and 8 mM ascorbic acid, in distilled H 2 O) for 1 h. 5 Reactions were quenched with 4 X SDS-PAGE loading buffer (200 mM Tris-HCl, pH 6.8, 40 % glycerol, 8 % sodium dodecyl sulphate, 0.4 % Bromophenol Blue, supplemented with 25 mM dithiothreitol) and samples were heated for 5 min at 95 °C, resolved by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting with a rat anti-HA antibody to detect ABHD16A and ABHD12 (Roche, clone 3F10).Infrared fluorescence was detected using an Odyssey imaging system (LI-COR, Inc).Percentage inhibition was quantified by measuring the difference in the intensity of the Azido-FP band relative to a DMSO control (no compound) and normalized to the HA signal using Image Studio software (LI-COR, Inc).No unexpected or unusually high safety hazards were encountered.

Molecular modeling
To evaluate the binding capability and selectivity of the abietane database from this study towards the human ABHD12 and ABHD16A proteins, we have performed molecular docking calculations using Autodock 4.2. 6e to the unavailability of any experimentally determined structure from both these proteins, we have used in this work the available AlphaFold 7 models with reference AF-A0A1A8MD55-F1, and AF-Q8N2K0-F1, respectively corresponding to ABHD16A and ABHD12 proteins.The 3D configurations of all evaluated compounds were generated using the program Balloon 8 to mol2 file format.The compounds and protein models pdbqt file format required to run all molecular docking calculations were generated using respectively, the prepare_ligand4.pyand the prepare_receptor4.pyscripts from AutoDockTools software package (version 1.5.7). 6While all compounds were set to be fully flexible, the protein models were set to their fixed configurations.The affinity maps for the atom types found on all dataset compounds were calculated using the program autogrid found in Autodock 4.2 software package.After aligning both structure models based on the catalytic triad positions (composed by a serine, histidine and as aspartate residue), a simulation box was centered on the 3D geometry center space coordinate determined from the positions of these residues.The search space was defined to 66, 70, and 80 points of dimension (with a spacing of 0.375 Angstroms) on the X, Y, and Z axis, to assure that the entire surface of interest was fully covered.In each docking assay, 1000 different poses were generated using Autodock's implemented genetic algorithm.The number of generations was set to 270000, while the size of populations was set to 500.All the remaining parameters in the search mode were set to their default.For each calculation, all generated poses were clustered with a 1.5 Angstrom cut-off.The lowest energy solution of the top-ranking cluster was selected as the most representative docking solution for each compound on each evaluated structural model.

1 H
NMR and 13 C NMR spectra were recorded in either CDCl 3 , CD 3 OD or DMSO-d 6 in a Bruker Ascend 400 spectrometer.The chemical shifts are reported in ppm relative to residual CHCl 3 (δ 7.26), CHD2OD (δ 3.31) or DMSO-d 5 (2.50) for 1 H NMR. For the 13 C NMR spectra, CDCl 3 (δ 77.16), CD 3 OD (δ 49.00) or DMSO-d 6 (39.52) were used as the internal standards.The coupling constants J are quoted in hertz (Hz).LC-MS analyses were executed with Waters Acquity® UPLC system (Waters, Milford MA, USA) with Acquity PDA detector and Waters Synapt G2 HDMS mass spectrometer (Waters, Milford MA, USA) via an ESI ion source.Samples were analyzed in positive, resolution ion mode.Mass range was set from 100 to 600.Separation was performed in Acquity UPLC® BEH C18 column (1.7 μm, 50 mm × 2.1 mm, Waters, Ireland) in 40 °C.The mobile phase consisted of 0.1% formic acid both in (A) H 2 O and (B) acetonitrile (Chromasolv® grade, Sigma-Aldrich, Steinheim, Germany).A linear gradient started at 95% of A and decreased to 10%.Purity of the biologically evaluated compounds was >95%, determined by the UPLC.No unexpected or unusually high safety hazards were encountered.

Figure S1 .
Figure S1.Inhibition of murine ABHD16A by competitive ABPP.HA-tagged mABHD16A enriched from HEK293T total membrane proteomes was incubated with compounds at the indicated concentrations followed by labelling with FP-azide and conjugation by click chemistry to an alkyne-infrared 800 dye (AK-IR800).Anti-HA primary antibody and an anti-rat IR680 secondary antibody were used to detect ABHD16A (IR680).mABHD16A inhibition was calculated by measuring the difference in FP-azide incorporation relative to DMSO control (-), normalized to protein levels.100 μM Palmostatin B (PB) was used as a positive control.A. Representative immunoblot images are shown: click chemistry signal (Top, AK-IR800), HA (Middle, IR680), and merge (Bottom).The position of molecular weight standards is shown on the left.In the middle panel, the same immunoblots are shown but with one lane removed for clarity.B. Bar chart showing mean percentage mABHD16A inhibition.Individual bullet points represent independent experiments.Error bars represent ± SEM.Statistical significance was determined by one-way ANOVA with Dunnett's post hoc test.For clarity, only statistically significant analysis is shown.*p<0.05;**p<0.01;***p< 0.001; ****p<0.0001.

Figure S2 .
Figure S2.Structures of S42 and S43.The preparation of these 12-thiazole abietanes has been reported before.9

Figure S3 .
Figure S3.ABHD12 (A) and ABHD16A (B) protein model representations with the catalytic triad highlighted in yellow and cyan sticks respectively, and all the dataset compounds docking solutions represented as coloured sticks.