Sensitive LC-MS/MS Method for the Quantification of Macrocyclic Gαq Protein Inhibitors in Biological Samples.

Markus Kuschak,Michaela Matthey,Gabriele M König,Marion Schneider,Daniela Wenzel,Jonathan G Schlegel,Christa E Müller,Alexander Seidinger,Stefan Kehraus,Bernd K Fleischmann,Jan H Voss

doi:10.3389/fchem.2020.00833

Markus Kuschak, Michaela Matthey + Show 9 more

Open Access

https://doi.org/10.3389/fchem.2020.00833

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Abstract

The cyclic depsipeptide FR900359 (FR) isolated from the plant Ardisia crenata and produced by endosymbiotic bacteria acts as a selective Gq protein inhibitor. It is a powerful tool to study G protein-coupled receptor signaling, and has potential as a novel drug for the treatment of pulmonary diseases and cancer. For pharmacokinetic studies, sensitive quantitative measurements of drug levels are required. In the present study we established an LC-MS/MS method to detect nanomolar concentrations of FR and the structurally related natural product YM-254890 (YM) in biological samples. HPLC separation coupled to ESI-QTOF-MS and UV-VIS detection was applied. For identification and quantification, the extract ion chromatogram (EIC) of M+1 was evaluated. Limits of detection (LOD) of 0.53–0.55 nM and limits of quantification (LOQ) of 1.6–1.7 nM were achieved for both FR and YM. This protocol was subsequently applied to determine FR concentrations in mouse organs and tissues after peroral application of the drug. A three-step liquid-liquid extraction protocol was established, which resulted in adequate recovery rates of typically around 50%. The results indicated low peroral absorption of FR. Besides the gut, highest concentrations were determined in eye and kidney. The developed analytical method will be useful for preclinical studies to evaluate these potent Gq protein inhibitors, which may have potential as future drugs for complex diseases.

Highlights

The structurally related natural macrocyclic depsipeptides, YM-254890 (YM) and FR900359 (FR, see Figure 1 for structures) have been described as potent and selective inhibitors of Gq proteins (Takasaki et al, 2004; Schrage et al, 2015)
Macrocyclic Gq Protein Inhibitor Analysis crenata reveals that secondary metabolism may be a key function for the Ardisia crenata leaf nodule symbiosis (Carlier et al, 2016)
We developed a three-step liquid-liquid extraction (LLE) method along with an LC-MS/MS protocol to detect and quantify nanomolar concentrations of FR and YM in mouse tissues and blood serum

Summary

Introduction

The structurally related natural macrocyclic depsipeptides, YM-254890 (YM) and FR900359 (FR, see Figure 1 for structures) have been described as potent and selective inhibitors of Gq proteins (Takasaki et al, 2004; Schrage et al, 2015). Macrocyclic Gq Protein Inhibitor Analysis crenata reveals that secondary metabolism may be a key function for the Ardisia crenata leaf nodule symbiosis (Carlier et al, 2016). The structure of both depsipeptides differs only in two residues, resulting in a higher lipophilicity of FR as compared to YM: FR contains a propionyl instead of an acetyl residue and an isopropyl instead of a methyl group (Figure 1, highlighted in gray circles). Inhibition of Gq signaling has been investigated in several disease models, and FR in particular, due to its long residence time (Kuschak et al, 2020), was found to be a promising drug candidate for the treatment of complex diseases such as asthma (Matthey et al, 2017), hypertension (Meleka et al, 2019), metabolic syndrome (Klepac et al, 2016), and cancers (Onken et al, 2018; Annala et al, 2019; Lapadula et al, 2019)

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