Multiple Target Monitoring by Label-Free Binding by Mass Spectrometry Directly In Tissue For Translational In Vitro Safety-Tox Studies

Polypharmacology describes the activity of compounds at multiple targets. Current research focuses on two aspects of polypharmacology: firstly, unintended polypharmacology can lead to adverse effects, and secondly, polypharmacology across several disease-relevant targets can improve therapeutic efficacy, prevent drug resistance, or reduce therapeutic target-related adverse effects.

In the drug development process, binding assays play a critical role in the assessment of the affinity of test compounds to specific targets. Traditionally, binding assays are performed using either radioactive, fluorescent, or luminescence labeled ligands; the “gold standard” being the use of radioligands. This latter approach does not allow for the monitoring of multiple targets simultaneously in complex biological matrices. Our aim was to determine the feasibility of using label-free multiple target monitoring (MTM) by Mass Spectrometry (MS) to analyze 10 receptors simultaneously directly in tissues.

The membrane fraction of a rat brain cortex was isolated, and for each of the 10 receptors a specific well-documented ligand selected. The quantification of each of the ligands individually or in a mixture was optimized by ultra-high performance LC-MS. For the binding assays, the methodology was identical to that used for radioligand binding assays. The IC50’s of each test compounds was determined for each of the 10 targets individually or simultaneously.

87% of the test compounds were found to have a binding affinity to at least one of the 10 targets. For the closely related α1 and α2 adrenergic receptors only phentolamine, a synthetic imidazoline, was found to bind to both receptors with a high affinity, whereas another 5 compounds demonstrated selectively to α1 over α2. A high level of correlation of IC₅₀ values were established between the individual binding by mass and the MTM binding for 27 targets with an IC₅₀ values below 11 µM.

The discriminatory power of MS enables MTM to evaluate target specificity in either disease or animal knockout models in complex biological matrices, opening new avenues in efficient in vitro translational drug discovery to meet challenges related to polypharmacology.