Identification of small molecule ceramide kinase inhibitors using a homogeneous chemiluminescence high throughput assay.

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.

In vitro ADME profiling using high-throughput rapidfire mass spectrometry: cytochrome p450 inhibition and metabolic stability assays.

Early assessment of absorption, distribution, metabolism, and excretion (ADME) properties of drug candidates has become an essential component of modern drug discovery. ADME characterization is important in identifying compounds early that are likely to fail in later clinical development because of suboptimal pharmacokinetic properties or undesirable drug-drug interactions. Proper utilization of ADME results, meanwhile, can prioritize candidates that are more likely to have good pharmacokinetic properties and also minimize potential drug-drug interactions. By integrating a RapidFire system with an API4000 mass spectrometer (RF-MS), we have established a high-throughput capability to profile compounds (>100 compounds/wk) in a panel of ADME assays in parallel with biochemical and cellular characterizations. Cytochrome P450 inhibition and time-dependent inhibition assays and microsomal stability assays were developed and fully optimized on the system. Compared with the classic liquid chromatography-mass spectrometry method, the RF-MS system generates consistent data with approximately 20-fold increase in throughput. The lack of chromatographic separation of compounds, substrates, and metabolites can complicate data interpretation, but this occurs in a small number of cases that are readily identifiable. Overall, this system has enabled a real-time and quantitative measurement of a large number of ADME samples, providing a rapid evaluation of clinically important drug-drug interaction potential and drug metabolic stability.

Mechanistic studies with potent and selective inducible nitric-oxide synthase dimerization inhibitors.

A series of potent and selective inducible nitric-oxide synthase (iNOS) inhibitors was shown to prevent iNOS dimerization in cells and inhibit iNOS in vivo. These inhibitors are now shown to block dimerization of purified human iNOS monomers. A 3H-labeled inhibitor bound to full-length human iNOS monomer with apparent Kd approximately 1.8 nm and had a slow off rate, 1.2 x 10(-4) x s(-1). Inhibitors also bound with high affinity to both murine full-length and murine oxygenase domain iNOS monomers. Spectroscopy and competition binding with imidazole confirmed an inhibitor-heme interaction. Inhibitor affinity in the binding assay (apparent Kd values from 330 pm to 27 nm) correlated with potency in a cell-based iNOS assay (IC50 values from 290 pm to 270 nm). Inhibitor potency in cells was not prevented by medium supplementation with l-arginine or sepiapterin, but inhibition decreased with time of addition after cytokine stimulation. The results are consistent with a mechanism whereby inhibitors bind to a heme-containing iNOS monomer species to form an inactive iNOS monomer-heme-inhibitor complex in a pterin- and l-arginine-independent manner. The selectivity for inhibiting dimerization of iNOS versus endothelial and neuronal NOS suggests that the energetics and kinetics of monomer-dimer equilibria are substantially different for the mammalian NOS isoforms. These inhibitors provide new research tools to explore these processes.

2-(Aminomethyl)-benzamide-based glycine transporter type-2 inhibitors.

Structure-activity studies on benzamide 1 obtained from library screening led to the discovery of a novel series of potent and selective glycine transporter type-2 inhibitors.