Recurrent genetic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) have been identified in multiple tumor types. The most frequent mutation, IDH1 R132H, is a gain-of-function mutation resulting in an enzyme-catalyzing conversion of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG). A high-throughput assay quantifying consumption of NADPH by IDH1 R132H has been optimized and implemented to screen 3 million compounds in 1536-well formats. The primary high-throughput screening hits were further characterized by RapidFire-mass spectrometry measuring 2-HG directly. Multiple distinct chemotypes were identified with nanomolar potencies (6-300 nM). All inhibitors were found to be inactive against the wild-type IDH1 homodimers. An IDH1 heterodimer between wild-type and R132H mutant is capable of catalyzing conversion of α-KG to 2-HG and isocitrate to α-KG. Interestingly, one of the inhibitors, EXEL-9324, was found to inhibit both conversions by the IDH1 heterodimer. This indicates the R132H/WT heterodimer may adopt conformations distinct from that of the R132H/R132H homodimer. Further enzymatic studies support this conclusion as the heterodimer exhibited a significantly lower apparent Michaelis-Menten constant for α-KG (K(m)=110 µM) compared with the R132H homodimer (K(m)= 1200 µM). The enhanced apparent affinity for α-KG suggests R132H/WT heterodimeric IDH1 can produce 2-HG more efficiently at normal intracellular levels of α-KG (approximately 100 µM).

Lipid kinases have emerged as potentially important therapeutic targets in oncology and inflammation. Ceramide kinase (CERK) is a lipid kinase that catalyzes the formation of ceramide-1-phosphate from ceramide, a sphingolipid that is a key mediator of cellular apoptosis. Ceramide-1-phosphate has been shown to enhance the production of pro-inflammatory eicosonoids, to promote cell proliferation, and potentially to reduce intracellular ceramide levels by inhibition of acidic sphingomyelinases. Here we describe a homogeneous chemiluminescence assay that directly measures the ceramide-dependent ATP depletion by recombinant full-length human CERK. As compared to reported CERK assays that have limitations on compound throughput, the chemiluminescence assay has been miniaturized to a 1,536-well microtiter plate format and utilized to screen an ultra-large compound library (>4 million compounds). Multiple chemical scaffolds have been identified as CERK kinase inhibitors and characterized mechanistically, which to our knowledge represent the first known small molecule CERK inhibitors with nanomolar activities. These compounds can serve as tools to further elucidate the CERK pathway and its role in ceramide metabolism and human diseases.