New Platform for Label-Free, Proximal Cellular Pharmacodynamic Assays: Identification of Glutaminase Inhibitors Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry

Cellular
pharmacodynamic assays are crucial aspects of lead optimization
programs in drug discovery. These assays are sometimes difficult to
develop, oftentimes distal from the target and frequently low throughput,
which necessitates their incorporation in the drug discovery funnel
later than desired. The earlier direct pharmacodynamic modulation
of a target can be established, the fewer resources are wasted on
compounds that are acting via an off-target mechanism. Mass spectrometry
is a versatile tool that is often used for direct, proximal cellular
pharmacodynamic assay analysis, but liquid chromatography-mass spectrometry
methods are low throughput and are unable to fully support structure–activity
relationship efforts in early medicinal chemistry programs. Infrared
matrix-assisted laser desorption electrospray ionization (IR-MALDESI)
is an ambient ionization method amenable to high-throughput cellular
assays, capable of diverse analyte detection, ambient and rapid laser
sampling processes, and low cross-contamination. Here, we demonstrate
the capability of IR-MALDESI for the detection of diverse analytes
directly from cells and report the development of a high-throughput,
label-free, proximal cellular pharmacodynamic assay using IR-MALDESI
for the discovery of glutaminase inhibitors and a biochemical assay
for hit confirmation. We demonstrate the throughput with a ∼100,000-compound
cellular screen. Hits from the screening were confirmed by retesting
in dose–response with mass spectrometry-based cellular and
biochemical assays. A similar workflow can be applied to other targets
with minimal modifications, which will speed up the discovery of cell
active lead series and minimize wasted chemistry resources on off-target
mechanisms.