Development and Optimization of Piperidyl-1,2,3-Triazole Ureas as Selective Chemical Probes of Endocannabinoid Biosynthesis

Hsu, Ku‐Lung; Tsuboi, Kazuma; Whitby, Landon R.; Speers, Anna E; Pugh, Holly; Inloes, Jordon M.; Cravatt, Benjamin F.

doi:10.1021/jm400898x

Cited by 54 publications

(64 citation statements)

References 30 publications

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“…3 C and G) displayed greater inhibitory potency at DAGLα than DAGLβ. Both compounds exhibited good selectivity against other serine hydrolases including MAGL and FAAH but showed cross-reactivity with ABHD6, as expected (20)(21)(22). To identify appropriate negative control probes, we confirmed that the ABHD6-selective inhibitors KT185 (Fig.…”

Section: Resultssupporting

confidence: 66%

Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure

Buczynski

Herman

Hsu

et al. 2017

Alcohol

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Section: Resultssupporting

confidence: 66%

Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure

Buczynski

Herman

Hsu

et al. 2017

Alcohol

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“…S1C and Dataset S1), whereas the 40+ other serine hydrolases detected by ABPP were unchanged. Selective loss of DDHD2 in DDHD2 −/− mice was further verified using DDHD2-directed (HT-01) and broad-spectrum [FP-rhodamine (FP-Rh)] fluorescent activity-based probes (17), where the former probe revealed the absence of a characteristic 75-kDa DDHD2 doublet (9) in DDHD2 −/− brains, whereas other serine hydrolase signals were unaltered (Fig. 1C).…”

Section: Resultsmentioning

confidence: 78%

“…S11A). Higher resolution, quantitative MS analyses using the ABPP-stable isotope labeling by amino acids in cell culture method (17) confirmed that KLH45 (25 nM, 4 h) completely inactivated DDHD2 (>95% inhibition) in Neuro2A cells and showed no cross-reactivity with any of the other 40+ detected serine hydrolases with the exception of ABHD6 (SI Appendix, Fig. S11B and Dataset S2).…”

Section: Triglyceride (Tag) Accumulation In Ddhd2mentioning

confidence: 83%

“…To ascertain whether brain TAG accumulation in DDHD2 −/− mice was due to loss of catalytic function of DDHD2, we sought to develop a selective inhibitor of this enzyme for in vivo pharmacological studies. We previously discovered that acyclic phenethyl-1,2,3-triazole ureas, including the HT-01 probe, react with DDHD2 in a covalent, irreversible manner (17). A survey of analogs of HT-01 revealed that KLH45 bearing a cyclohexyl group in place of the extended BODIPY fluorophore of HT-01 (Fig.…”

Section: Triglyceride (Tag) Accumulation In Ddhd2mentioning

confidence: 99%

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The hereditary spastic paraplegia-related enzyme DDHD2 is a principal brain triglyceride lipase

Inloes

Hsu

Dix

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Complex hereditary spastic paraplegia (HSP) is a genetic disorder that causes lower limb spasticity and weakness and intellectual disability. Deleterious mutations in the poorly characterized serine hydrolase DDHD2 are a causative basis for recessive complex HSP. DDHD2 exhibits phospholipase activity in vitro, but its endogenous substrates and biochemical functions remain unknown. Here, we report the development of DDHD2 −/− mice and a selective, in vivo-active DDHD2 inhibitor and their use in combination with mass spectrometry-based lipidomics to discover that DDHD2 regulates brain triglycerides (triacylglycerols, or TAGs). DDHD2 −/− mice show age-dependent TAG elevations in the central nervous system, but not in several peripheral tissues. Large lipid droplets accumulated in DDHD2 −/− brains and were localized primarily to the intracellular compartments of neurons. These metabolic changes were accompanied by impairments in motor and cognitive function. Recombinant DDHD2 displays TAG hydrolase activity, and TAGs accumulated in the brains of wild-type mice treated subchronically with a selective DDHD2 inhibitor. These findings, taken together, indicate that the central nervous system possesses a specialized pathway for metabolizing TAGs, disruption of which leads to massive lipid accumulation in neurons and complex HSP syndrome. D etermining the genetic basis for rare hereditary human diseases has benefited from advances in DNA sequencing technologies (1). As a greater number of disease-causing mutations are mapped, however, it is also becoming apparent that many of the affected genes code for poorly characterized proteins. Assigning biochemical and cellular functions to these proteins is critical to achieve a deeper mechanistic understanding of human genetic disorders and for identifying potential treatment strategies.Hereditary spastic paraplegia (HSP) is a genetically heterogeneous neurologic syndrome marked by spasticity and lower extremity weakness (2). Many genetic types of HSP have been identified and are numbered according to their order of discovery [spastic paraplegia (SPG) 1-72] (2, 3). Of these genetic variants, more than 40 have been mapped to causative mutations in protein-coding genes. HSP genes code for a wide range of proteins that do not conform to a single sequence-or function-related class. A subset of HSP genes, including PNPLA6 (or neuropathytarget esterase) (SPG39) (4), DDHD1 (SPG28) (5), and DDHD2 (SPG54) (3, 6-8), code for serine hydrolases. These enzymes have been designated as (lyso)phospholipases based on in vitro substrate assays (9-11), but their endogenous substrates and physiological functions remain poorly understood. The mutational landscape that affects these lipid hydrolases to cause recessive HSP is complex but collectively represents a mix of null and putatively null and/or functional mutations. Moreover, the type of HSP appears to differ in each case, with DDHD1 mutations causing uncomplicated HSP, whereas PNPLA6 and DDHD2 mutations lead to complex forms of the disease that...

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“…A thin layered chromatography based radio-assay involving radio-labelled DAG has also been utilized to measure DAGL activity 2,28 . Several fluorescently labelled activity based probes with applications in gel based assays have been developed for the DAGLs, ranging from the generic serine hydrolase binding probe -fluorophosphonate 27 to more recent probes with ever improving selectivity for DAGLα and DAGLβ 13, 29,30 . Recently, higher throughput assays suitable for drug-discovery programs for the DAGLs have been reported.…”

Section: Introductionmentioning

confidence: 99%

Assay and Inhibition of the Purified Catalytic Domain of Diacylglycerol Lipase Beta

Singh

Markwick

et al. 2016

Biochemistry

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The diacylglycerol lipases (DAGLα and DAGLβ) hydrolyse DAG to generate 2-arachidonoylglycerol (2-AG), the principal endocannabinoid and main precursor of arachidonic acid (AA). The DAGLs make distinct tissue specific contributions towards 2-AG and AA levels and therefore selective modulators for these enzymes could play crucial roles towards harnessing their therapeutic potential. Relatively high-throughput assays have recently been reported for DAGLα and have proven useful towards the characterization of inhibitors of this enzyme. Similar assays are also warranted for DAGLβ which was the aim of this study. We first adapted previously reported DAGLα membrane assays (using PNPB and DiFMUO as substrates) to measure recombinant DAGLβ activity in membranes. In contrast to results with DAGLα, both substrates provided a relatively limited signal window for measuring DAGLβ activity, however, an improved window was obtained when employing a third commercially available substrate, EnzChek. In order to further improve on the assay parameters, we successfully purified the glutathione S-transferase (GST) tagged catalytic domain of DAGLβ. Activity of the enzyme was confirmed using EnzChek as well as two DAGL inhibitors . The purified DAGLβ catalytic domain assay described here provides the basis for a relatively clean and convenient assay with the potential to be adapted for high-throughput drug discovery efforts.

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Development and Optimization of Piperidyl-1,2,3-Triazole Ureas as Selective Chemical Probes of Endocannabinoid Biosynthesis

Cited by 54 publications

References 30 publications

Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure

Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure

The hereditary spastic paraplegia-related enzyme DDHD2 is a principal brain triglyceride lipase

Assay and Inhibition of the Purified Catalytic Domain of Diacylglycerol Lipase Beta

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