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Development and implementation of novel mass spectrometric methods for metabolism studies in drug discoveries
No full textThe field of drug discovery has evolved significantly over the past few decades, with a growing emphasis on understanding the metabolic pathways and biotransformation processes that influence the pharmacokinetics and pharmacodynamics of drug candidates. Novel mass spectrometric methods have emerged as powerful tools in this domain, facilitating the comprehensive analysis of metabolic profiles and enabling the identification of metabolites with implications for safety, efficacy, and overall drug development. However, challenges remain in quantifying metabolic flux—an essential aspect of understanding drug action—particularly due to the limitations of traditional isotope kinetic assays, which often involve lengthy sample preparation and the use of radioactive tracers. Furthermore, the synthesis of drug metabolites, which frequently possess complex structures, is vital for evaluating the metabolism, pharmacokinetic properties, and safety profiles of drug candidates. These synthetic processes are often cumbersome and may require multiple-step de novo synthetic routes.
Accordingly, the goal of this work is to develop innovative MS-based methods for bioanalysis and metabolite synthesis. Three projects are included with developing two novel technologies, i.e., online electrospray microdroplet derivatization and electrochemical mass spectrometry, showing their unique applications in metabolism studies for drug discovery. Firstly, microdroplet reaction is employed to facilitate on-the-fly derivatization during the electrospray ionization process. This method allows for rapid, sensitive measurement of glucose homeostasis. Using this technique, Semaglutide\u27s effects on endogenous 12C-glucose and exogenous [U-13C6]glucose in plasma and Pioglitazone-mediated changes in glucose uptake in mouse hearts were elucidated. These findings demonstrated the potential of microdroplet-based bioanalysis to accelerate metabolic flux studies and improve drug discovery workflows.
Secondly, a one-step Shono-type electrochemical oxidation was employed for the synthesis of chiral a-hydroxy amide metabolites for the orexin receptor antagonists MK-8133 and MK-6096. Reaction screening on a custom-built electrochemistry/mass spectrometry (EC/MS) platform enabled efficient optimization of conditions at pico- to nano-mole scales. The oxidation, conducted in aqueous media, produced a-piperidinols with exclusive regio- and stereoselectivity, confirmed by high-resolution NMR. Density functional theory (DFT) calculations revealed that regioselectivity was driven by favorable transition state energetics, while stereoselectivity was dictated by steric effects in the U-shaped cation intermediate.
Thirdly, a stable isotope tracer protocol utilizing deuterium-labeled water was developed to quantify protein flux in mouse plasma using LC-MS/MS. Unlike typical LC-MS/MS workflows that require time-consuming immunodepletion, this approach successfully quantified the flux of three proteins (albumin, apoprotein A1, and E) using only 5µL of plasma. The ability to quantify the incorporation of deuterium into plasma proteins provided valuable insights into novel protein targets and disease progression.
The successful application of these emerging technologies addressed critical bottlenecks in drug bioanalysis and metabolite synthesis, offering high throughput and cost-efficient studies of metabolism in biopharmaceutical settings
