HTS MALDI-TOF MDD · MALDI-TOF mass spectrometry metabolite screening assays for drug discovery in human disease

MALDI-TOF mass spectrometry metabolite screening assays for drug discovery in human disease

Discovery for drug targets is a key step within the process of drug development, and is the corner stone in the pharmaceutical industry. This is mostly achieved by high-throughput screening (HTS) approaches in which a large number of chemical substances are assayed for a specific effect or activity in diverse areas of biology. Mass spectrometry (MS) has become a widely adopted tool in this field as it offers the possibility to simultaneously track molecules in a label-free manner, provides excellent signal to noise, reproducibility, assay precision, and a significantly reduced reagent cost when compared to fluorescence-based assays. Matrix-assisted laser/desorption ionisation time of flight (MALDI-TOF) is the most validated surface ionisation method for HTS approaches, but reported applications of this technology have been limited to in vitro assays with simple readouts and to peptide/protein-centric activity assays. To date, comprehensive and unbiased metabolomics based HTS approaches for cellular assays with MALDI-TOF have not been explored. The objective of this fellowship was to set up a MALDI-TOF based cellular assay for HTS metabolomics drug discovery in order to identify a set of metabolites to screen which will enable us to unbiasly and comprehensibly measure, in a HT manner, how drugs affect different metabolic pathways while simultaneously mapping the metabolic profile of the cell. Originally, the plan was to: 1. Develop a robust protocol for the detection, identification, and quantification of ~30-50 small metabolites for cellular assays. 2. Apply this protocol to track metabolites in a pulmonary fibrosis cellular model, as well as in fibrotic lung tissue sections using MS imaging. 3. Implement this platform in Boehringer Ingelheim in Germany (3-month secondment). Conclusion of action: First, a robust MALDI-TOF MS protocol was developed for the detection, identification, and quantification of small metabolites and proteins for cellular assays. Different matrices, extraction procedures, and derivatization agents were tested. Moreover, a data analysis pipeline, including bioinformatic and machine learning tools, was developed for the analysis. Then, this protocol was applied to track the ‘fingerprint’ of different biomarkers in different idiopathic pulmonary fibrosis (IPF) cellular models, including primary cells from IPF donors. The MALDI cellular assay developed is based on the concept that metabolites, lipids, and proteins that ionise in MALDI provide a specific fingerprint, depending on the cellular state. Comparing healthy and disease conditions allows for the identification of biomarkers which not only provides insights to the disease but also can serve as readouts for drug screen. With bioinformatic tools, readouts can be generated to distinguish treatments with the aim that cells treated with a ‘hit’ will reverse to the healthy phenotype. A small library screen using the OpnMe compounds from Boehringer Ingelheim was conducted at Newcastle University. Unfortunately, we did not reach the stage of finding and confirming ‘hit’ compounds using MALDI-MSI. Therefore, we decided to move towards proteomics to understand the system and get some insight of how proteins are affected using our cell model and treatment. Moreover, because of the pandemic and due to travel-restrictions, the 3-month industrial secondment to Boehringer Ingelheim did not happen.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз