Phospholipase Cγ2 regulates endocannabinoid and eicosanoid networks in innate immune cells.

Human genetic studies have pointed to a prominent role for innate immunity and lipid pathways in immunological and neurodegenerative disorders. Our understanding of the composition and function of immunomodulatory lipid networks in innate immune cells, however, remains incomplete. Here, we show that phospholipase Cγ2 (PLCγ2 or PLCG2)-mutations in which are associated with autoinflammatory disorders and Alzheimer's disease-serves as a principal source of diacylglycerol (DAG) pools that are converted into a cascade of bioactive endocannabinoid and eicosanoid lipids by DAG lipase (DAGL) and monoacylglycerol lipase (MGLL) enzymes in innate immune cells. We show that this lipid network is tonically stimulated by disease-relevant human mutations in PLCγ2, as well as Fc receptor activation in primary human and mouse macrophages. Genetic disruption of PLCγ2 in mouse microglia suppressed DAGL/MGLL-mediated endocannabinoid-eicosanoid cross-talk and also caused widespread transcriptional and proteomic changes, including the reorganization of immune-relevant lipid pathways reflected in reductions in DAGLB and elevations in PLA2G4A. Despite these changes, Plcg2-/- mice showed generally normal proinflammatory cytokine and chemokine responses to lipopolysaccharide treatment, instead displaying a more restricted deficit in microglial activation that included impairments in prostaglandin production and CD68 expression. Our findings enhance the understanding of PLCγ2 function in innate immune cells, delineating a role in cross-talk with endocannabinoid/eicosanoid pathways and modulation of subsets of cellular responses to inflammatory stimuli.

ABHD17 regulation of plasma membrane palmitoylation and N-Ras-dependent cancer growth.

Multiple Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase inhibitors such as Palmostatin M (Palm M) block N-Ras depalmitoylation, but lack specificity and target several enzymes displaying depalmitoylase activity. Here, we describe ABD957, a potent and selective covalent inhibitor of the ABHD17 family of depalmitoylases, and show that this compound impairs N-Ras depalmitoylation in human acute myeloid leukemia (AML) cells. ABD957 produced partial effects on N-Ras palmitoylation compared with Palm M, but was much more selective across the proteome, reflecting a plasma membrane-delineated action on dynamically palmitoylated proteins. Finally, ABD957 impaired N-Ras signaling and the growth of NRAS-mutant AML cells in a manner that synergizes with MAP kinase kinase (MEK) inhibition. Our findings uncover a surprisingly restricted role for ABHD17 enzymes as regulators of the N-Ras palmitoylation cycle and suggest that ABHD17 inhibitors may have value as targeted therapies for NRAS-mutant cancers.

AIG1 and ADTRP are endogenous hydrolases of fatty acid esters of hydroxy fatty acids (FAHFAs) in mice.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of signaling lipids with anti-inflammatory and anti-diabetic properties. However, the endogenous regulation of FAHFAs remains a pressing but unanswered question. Here, using MS-based FAHFA hydrolysis assays, LC-MS-based lipidomics analyses, and activity-based protein profiling, we found that androgen-induced gene 1 (AIG1) and androgen-dependent TFPI-regulating protein (ADTRP), two threonine hydrolases, control FAHFA levels in vivo in both genetic and pharmacologic mouse models. Tissues from mice lacking ADTRP (Adtrp-KO), or both AIG1 and ADTRP (DKO) had higher concentrations of FAHFAs particularly isomers with the ester bond at the 9th carbon due to decreased FAHFA hydrolysis activity. The levels of other lipid classes were unaltered indicating that AIG1 and ADTRP specifically hydrolyze FAHFAs. Complementing these genetic studies, we also identified a dual AIG1/ADTRP inhibitor, ABD-110207, which is active in vivo Acute treatment of WT mice with ABD-110207 resulted in elevated FAHFA levels, further supporting the notion that AIG1 and ADTRP activity control endogenous FAHFA levels. However, loss of AIG1/ADTRP did not mimic the changes associated with pharmacologically administered FAHFAs on extent of upregulation of FAHFA levels, glucose tolerance, or insulin sensitivity in mice, indicating that therapeutic strategies should weigh more on FAHFA administration. Together, these findings identify AIG1 and ADTRP as the first endogenous FAHFA hydrolases identified and provide critical genetic and chemical tools for further characterization of these enzymes and endogenous FAHFAs to unravel their physiological functions and roles in health and disease.

Monoacylglycerol Lipase Inhibition in Human and Rodent Systems Supports Clinical Evaluation of Endocannabinoid Modulators.

Monoacylglycerol lipase (MGLL) is the primary degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). The first MGLL inhibitors have recently entered clinical development for the treatment of neurologic disorders. To support this clinical path, we report the pharmacological characterization of the highly potent and selective MGLL inhibitor ABD-1970 [1,1,1,3,3,3-hexafluoropropan-2-yl 4-(2-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-4-chlorobenzyl)piperazine-1-carboxylate]. We used ABD-1970 to confirm the role of MGLL in human systems and to define the relationship between MGLL target engagement, brain 2-AG concentrations, and efficacy. Because MGLL contributes to arachidonic acid metabolism in a subset of rodent tissues, we further used ABD-1970 to evaluate whether selective MGLL inhibition would affect prostanoid production in several human assays known to be sensitive to cyclooxygenase inhibitors. ABD-1970 robustly elevated brain 2-AG content and displayed antinociceptive and antipruritic activity in a battery of rodent models (ED50 values of 1-2 mg/kg). The antinociceptive effects of ABD-1970 were potentiated when combined with analgesic standards of care and occurred without overt cannabimimetic effects. ABD-1970 also blocked 2-AG hydrolysis in human brain tissue and elevated 2-AG content in human blood without affecting stimulated prostanoid production. These findings support the clinical development of MGLL inhibitors as a differentiated mechanism to treat pain and other neurologic disorders.

Inflammation causes insulin resistance in mice via interferon regulatory factor 3 (IRF3)-mediated reduction in FAHFA levels.

Obesity-induced inflammation causes metabolic dysfunction, but the mechanisms remain elusive. Here we show that the innate immune transcription factor interferon regulatory factor (IRF3) adversely affects glucose homeostasis through induction of the endogenous FAHFA hydrolase androgen induced gene 1 (AIG1) in adipocytes. Adipocyte-specific knockout of IRF3 protects male mice against high-fat diet-induced insulin resistance, whereas overexpression of IRF3 or AIG1 in adipocytes promotes insulin resistance on a high-fat diet. Furthermore, pharmacological inhibition of AIG1 reversed obesity-induced insulin resistance and restored glucose homeostasis in the setting of adipocyte IRF3 overexpression. We, therefore, identify the adipocyte IRF3/AIG1 axis as a crucial link between obesity-induced inflammation and insulin resistance and suggest an approach for limiting the metabolic dysfunction accompanying obesity.

Quantification of In Vivo Target Engagement Using Microfluidic Activity-Based Protein Profiling.

Accurate measurement of drug-target interactions in vivo is critical for both preclinical development and translation to clinical studies, yet many assays rely on indirect measures such as biomarkers associated with target activity. Activity-based protein profiling (ABPP) is a direct method of quantifying enzyme activity using active site-targeted small-molecule covalent probes that selectively label active but not inhibitor-bound enzymes. Probe-labeled enzymes in complex proteomes are separated by polyacrylamide gel electrophoresis and quantified by fluorescence imaging. To accelerate workflows and avoid imaging artifacts that make conventional gels challenging to quantify, we adapted protocols for a commercial LabChip GXII microfluidic instrument to permit electrophoretic separation of probe-labeled proteins in tissue lysates and plasma, and quantification of fluorescence (probe/protein labeling ratio of 1:1). Electrophoretic separation on chips occurred in 40 s per sample, and instrument software automatically identified and quantified peaks, resulting in an overall time savings of 3-5 h per 96-well sample plate. Calculated percent inhibition was not significantly different between the two formats. Chip performance was consistent between chips and sample replicates. Conventional gel imaging was more sensitive but required five times higher sample volume than microfluidic chips. Microfluidic chips produced results comparable to those of gels but with much lower sample consumption, facilitating assay miniaturization for scarce biological samples. The time savings afforded by microfluidic electrophoresis and automatic quantification has allowed us to incorporate microfluidic ABPP early in the drug discovery workflow, enabling routine assessments of tissue distribution and engagement of targets and off-targets in vivo.

Identification of ABX-1431, a Selective Inhibitor of Monoacylglycerol Lipase and Clinical Candidate for Treatment of Neurological Disorders.

The serine hydrolase monoacylglycerol lipase (MGLL) converts the endogenous cannabinoid receptor agonist 2-arachidonoylglycerol (2-AG) and other monoacylglycerols into fatty acids and glycerol. Genetic or pharmacological inactivation of MGLL leads to elevation in 2-AG in the central nervous system and corresponding reductions in arachidonic acid and eicosanoids, producing antinociceptive, anxiolytic, and antineuroinflammatory effects without inducing the full spectrum of psychoactive effects of direct cannabinoid receptor agonists. Here, we report the optimization of hexafluoroisopropyl carbamate-based irreversible inhibitors of MGLL, culminating in a highly potent, selective, and orally available, CNS-penetrant MGLL inhibitor, 28 (ABX-1431). Activity-based protein profiling experiments verify the exquisite selectivity of 28 for MGLL versus other members of the serine hydrolase class. In vivo, 28 inhibits MGLL activity in rodent brain (ED50 = 0.5-1.4 mg/kg), increases brain 2-AG concentrations, and suppresses pain behavior in the rat formalin pain model. ABX-1431 (28) is currently under evaluation in human clinical trials.