Identification of DGAT2 Inhibitors Using Mass Spectrometry

Abstract

Mass spectrometry offers significant advantages over other detection technologies in the areas of hit finding, hit validation, and medicinal chemistry compound optimization. The foremost obvious advantage is the ability to directly measure enzymatic product formation. In addition, the inherent sensitivity of the liquid chromatography/mass spectrometry (LC/MS) approach allows the execution of enzymatic assays at substrate concentrations typically at or below substrate K m . Another advantage of the LC/MS approach is the ability to assay impure enzyme systems that would otherwise be difficult to prosecute with traditional labeled methods. This approach was used to identify inhibitors of diacylglycerol O-acyltransferase-2 (DGAT2), a transmembrane enzyme involved in the triglyceride (TG) production pathway. The LC/ MS approach was employed because of its increased assay window (compared with control membranes) of more than sevenfold compared with less than twofold with a conventional fluorogenic assay. The ability to generate thousands of dose-dependent IC 50 data was made possible by the use of a staggered parallel LC/MS system with fast elution gradients. From the hit-deconvolution efforts, several structural scaffold series were identified that inhibit DGAT2 activity. Additional profiling of one chemotype in particular identified two promising reversible and selective compounds (compound 15 and compound 16) that effectively inhibit TG production in mouse primary hepatocytes.

DGAT2 Inhibition Alters Aspects of Triglyceride Metabolism in Rodents but Not in Non-human Primates

“…Cells were plated at ideal confluency: for 96-well, collagen-coated dishes this was 40K-50K/well for mouse hepatocytes, 50K for human hepatocytes and 60K for NHP hepatocytes. Cellular TG secretion was determined by LC-MS/MS (Song et al, 2016). Briefly, cells were maintained in culture medium as described above at 37 C with 5% CO 2 .…”

Small structural changes of the imidazopyridine diacylglycerol acyltransferase 2 (DGAT2) inhibitors produce an improved safety profile

“…Suppression of the DGAT2 isoform with ASO improved diet-induced hyperlipidemia, hepatic steatosis and insulin resistance in rodents. [16][17][18] A number of small molecule DGAT2 inhibitors from distinct chemical series have been reported in the literature, [19][20][21][22][23] and recently our group 24 and others 25,26 reported in vivo pharmacology results. Treatment of dyslipidemic rodents with the selective DGAT2 inhibitor PF-06424439 (compound 1) § caused a reduction in plasma cholesterol as well as plasma and hepatic TG levels.…”

High‐Throughput Bioassays using “Dip‐and‐Go” Multiplexed Electrospray Mass Spectrometry

“…[6] Fort hese reasons,m ass spectrometry (MS) is widely considered an attractive alternative to optical detection methods for HTS bioassays, [1b] due to its inherent selectivity,s ensitivity and label-free characteristics.A s ar esult, an umber of MS platforms have been applied to screening of bioassays,including LC-MS at rates of ca. 0.5 to 8min/sample, [7] RapidFire-MS at rates of ca. 6t o8s/sample, MALDI-MS at ar ate of ca.…”