Development of an Activity‐Based Probe and In Silico Design Reveal Highly Selective Inhibitors for Diacylglycerol Lipase‐α in Brain

Abstract

Diacylglycerol lipase-a (DAGL-a) is an intracellular, multidomain protein responsible for the formation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the central nervous system. [1] 2-AG is an endogenous signaling lipid that interacts with the cannabinoid CB1 and CB2 receptors. [2] Little is known about the regulation of its biosynthetic pathway and it is largely unclear to what extent 2-AG is responsible for distinct cannabinoid CB1 receptor mediated biological processes. Selective inhibitors of DAGL-a may contribute to a more fundamental understanding of the physiological role of 2-AG and may serve as potential drug candidates for the treatment of obesity and neurodegenerative diseases. [3] Currently, there are no selective inhibitors and activity-based probes available for the study of DAGL-a. [4] The identification of selective DAGL-a inhibitors is hampered by a lack of structural knowledge of the target, and a lack of assays that make use of endogenous DAGL-a activity in proteomes. No crystal structures are available and no homology models have been reported to aid hit identification and to guide optimization of the inhibitors. Determination of the selectivity of the inhibitors in native tissues is important because DAGL-a belongs to the serine hydrolase family, which contains more than 200 members with various physiological functions. [5] Fluorophosphonate (FP)based probes are routinely employed in competitive activitybased protein profiling (ABPP) experiments to determine the selectivity of serine hydrolase inhibitors in complex proteomes. DAGL-a, however, does not react with these activitybased probes. [6] Therefore, a new probe that can label native DAGL-a would be of value for studying the potency and selectivity of novel DAGL-a inhibitors in brain proteomes.Here we present a strategy that combines a knowledge-based in silico design approach and the development of a novel activity-based probe (ABP), based on the nonselective DAGL-a inhibitor tetrahydrolipstatin (THL; also known as Orlistat, a drug used for the treatment of obesity). This strategy resulted in the rapid identification of DAGL-a inhibitors with a new chemotype and high selectivity in the brain proteome.To identify novel DAGL-a inhibitors, we built a pharmacophore model based on THL using Discovery Studio Software Suite from Accelrys. Since THL can assume many different conformations, we searched the protein crystallographic database for crystal structures with a bioactive conformation for THL. A cocrystal structure of THL with fatty acid synthase (pdb-code: 2PX6) was identified (Figure 1 A) [7] that contains the same Ser-His-Asp catalytic triad and typical a/b hydrolase fold motif as DAGL-a. In this cocrystal structure, the nucleophilic Ser of the enzyme is covalently attached to the carbonyl moiety of the lactone. We reconstituted the ester to form the b-lactone to recover the active warhead of THL. After optimization of the geometry of the lactone, the resulting conformation was used to generate two pharmacophore models (Figure...

Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons

“…We investigated in detail the binding pose of LEI104 in DAGL-α using a molecular dynamics simulation in our previously generated homology model. 21 From this analysis we identified an additional hydrophobic pocket close to the catalytic site that did not appear to be occupied by LEI104 (Figure 1B). Introduction of a phenyl substituent at the 6-position of the oxazolopyridine would allow us to probe this pocket with the aim of increasing potency and/or selectivity.…”

“…24 LEI105 proved to be a potent inhibitor with a pIC 50 of 8.5 ± 0.06 (n=4), thus some 10-fold more potent than LEI104 (7.4 ± 0.05; n=4). 21 Reduction of the α–keto group to the corresponding alcohol (compound 8 ) led to a ~150 fold drop of activity against DAGL-α, thereby indicating that the α–carbonyl in LEI105 reacts with the active site serine hydroxyl to form a covalent, though reversible, enzyme-inhibitor hemiketal adduct. To confirm that LEI105 was also able to block conversion of the natural substrate 1-stearoyl-2-arachidonoyl- sn -glycerol of DAGL-α to the endocannabinoid 2-AG, we employed our recently developed real-time, fluorescence-based assay.…”

“…19 Previously, we have reported on the design, synthesis and characterization of a specific β-lactone-containing probe (MB064), which was tailor-made for the detection of DAGL-α activity. 20,21 Using this probe we identified 1-(oxazolo[4,5-b]pyridin-2-yl)-6-phenylhexan-1-one (LEI104, Figure 1), which belongs to the class of α-ketoheterocycles, as the first reversible inhibitor for DAGL-α. LEI104 was, however, weakly active in a cellular assay and was not selective over FAAH, the enzyme responsible for the metabolism of the other endocannabinoid anandamide (AEA).…”

Combining cross-metathesis and activity-based protein profiling: New β-lactone motifs for targeting serine hydrolases

“…These enzyme assignments were based on previous studies where gel-based ABPP experiments of mouse membrane proteome were related to mass spectrometry-based identifications of enzymes. 12b, 20 Profiles of the human COLO205 colon cancer membrane proteome also revealed a diverse set of serine hydrolase targets for the β-lactone probes (Figure 3B). …”

“…11 While this channel may not be a universal motif, we and others have found that THL interacts with a variety of serine hydrolases. 12 Böttcher and Sieber also took inspiration from the aliphatic chains of β-lactone natural products in the design of β-lactone ABPPs. 1e In the initial series, for ease of synthesis we elected to use a simple alkyl chain at C4 and chose the chain length based on nocardiolactone, which has the same number of carbons in its alkyl chain as THL, giving us the opportunity to develop the methodology around a straightforward synthesis of (±)-nocardiolactone.…”