Measuring Drug Metabolism Kinetics and Drug–Drug Interactions Using Self-Assembled Monolayers for Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry

Abstract

The competition of two drugs for the same metabolizing enzyme is a common mechanism for drug-drug interactions that can lead to altered kinetics in drug metabolism and altered elimination rates in vivo. With the prevalence of multidrug therapy, there is great potential for serious drug-drug interactions and adverse drug reactions. In an effort to prevent adverse drug reactions, the FDA mandates the evaluation of the potential for metabolic inhibition by every new chemical entity. Conventional methods for assaying drug metabolism (e.g., those based on HPLC) have been established for measuring drug-drug interactions; however, they are low-throughput. Here we describe an approach to measure the catalytic activity of CYP2C9 using the high-throughput technique self-assembled monolayers for matrix-assisted laser desorption-ionization (SAMDI) mass spectrometry. We measured the kinetics of CYP450 metabolism of the substrate, screened a set of drugs for inhibition of CYP2C9 and determined the Ki values for inhibitors. The throughput of this platform may enable drug metabolism and drug-drug interactions to be interrogated at a scale that cannot be achieved with current methods.

High-Throughput Affinity Selection Mass Spectrometry Using SAMDI-MS to Identify Small-Molecule Binders of the Human Rhinovirus 3C Protease

“…Second, there remains a need for high-throughput and robust screening assays suitable for identifying ligand–target interactions. Here we expand the capabilities of the SAMDI technology, extensively reported as a powerful label-free and high-throughput assay for measuring biochemical and chemical reactions, 9,18–28 to offer a high-throughput ASMS assay with key benefits over traditional ASMS approaches.…”

Label-Free Screening of SARS-CoV-2 NSP14 Exonuclease Activity Using SAMDI Mass Spectrometry

“…Moreover, the platform is amenable to any buffer system, including detergents, high salts, carrier proteins, organic additives, and even cell lysates. 19–21 SAMDI-MS has been reported to measure biochemical activities on peptides, 22–24 DNA, 25 and small molecules, 26–29 and has recently been validated on RNA substrates. 30 A major benefit of SAMDI-MS for RNA substrates is the ability to monitor the purity and integrity of RNA in every reaction well, where RNases present in most labs and/or enzyme preps can lead to RNA degradation and false positives/negatives in label-based approaches such as Forster resonance energy transfer.…”

Traceless Immobilization of Analytes for High-Throughput Experiments with SAMDI Mass Spectrometry

“…7,8,14−16 In either case, the substrate must be modified with a functional group that allows its immobilization. 16 While this requirement for an immobilization tag is often not problematic, there are cases where the introduction of the tag is either not straightforward or is incompatible with the activity to be measured. SAMDI has also been used in the discovery and study of reactions, and here too the need for a functional group can interfere with the intended reaction.…”

“…The use of an analogue of a drug that is modified to include an immobilization tag carries the risk that the functional group will alter the activity toward a metabolizing enzyme. 16 To demonstrate this application, we assayed hydroxylation of the drug tolbutamide by CYP2C9*1, a P450 liver enzyme. 16 The enzyme (0.4 μM), and tolbutamide (50 μM) were combined in 15 μL of tris buffer (100 mM, pH 7.5) with the NADPH-regenerating system (1.3 mM NADP + , 3.3 mM glucose-6-phosphate, 3.3 mM magnesium chloride, and 0.4 U/mL glucose-6-phosphate dehydrogenase) and allowed to react for 0 and 60 min at 37 °C.…”

“…16 To demonstrate this application, we assayed hydroxylation of the drug tolbutamide by CYP2C9*1, a P450 liver enzyme. 16 The enzyme (0.4 μM), and tolbutamide (50 μM) were combined in 15 μL of tris buffer (100 mM, pH 7.5) with the NADPH-regenerating system (1.3 mM NADP + , 3.3 mM glucose-6-phosphate, 3.3 mM magnesium chloride, and 0.4 U/mL glucose-6-phosphate dehydrogenase) and allowed to react for 0 and 60 min at 37 °C. The reactions were stopped with HCl (3 M, 5 μL) and proteins were removed by pelleting with high-speed centrifugation, followed by extraction of tolbutamide and hydroxy-tolbutamide with diethyl ether.…”