Periodic Reporting for period 1 - HTS MALDI-TOF MDD (MALDI-TOF mass spectrometry metabolite screening assays for drug discovery in human disease)
Período documentado: 2020-04-01 hasta 2022-03-31
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.
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.
WP1: The goal of this WP was to develop a novel cellular assay protocol for untargeted metabolomics MALDI-TOF MS. I started with A549 cells to optimize and find best sample preparation conditions for assay. Using these cells, I tested different matrices, additives, extraction procedures, and derivatization strategies to see which combination worked best. I ended up exploring various data analysis, bioinformatics tools, and machine learning strategies for data analysis because the data was complex. Then, I moved towards a more relevant model, using human nasal epithelial cells in air-liquid-interface, and small airway epithelial cells. With aide from machine learning strategies, I was able to grasp important metabolic features of cells in order to track this in a high-throughput manner.
Outcome: I presented this work (oral talks) at SLAS 2022 (virtually), ACS 2022 (virtually), and ASMS 2022 (in-person). Internally at Newcastle University, I presented this work twice in departmental seminars.
Publication: Marín-Rubio, J.L. Heap, R.E. Dueñas, M.E. Heunis, T., Inns, J., Scott, J., Simpson, A.J. Blair, H., Heidenreich, O., Allan, J.M. Saxty, B. and Trost, M.*, A MALDI-TOF assay identifies nilotinib as an inhibitor of inflammation in acute myeloid leukaemia and multiple myeloma, (2021) biorxiv
WP2:
Once I had the model system validated, I worked on identifying potential fibrotic mediators in primary culture model in a high-throughput manner. I conducted a screening with known inhibitors first to see if that assay was working, followed by library screen using the OpnMe compounds from Boehringer Ingelheim.
WP3:
Unfortunately, we are not in 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. During this time, I developed my expertise in proteomics and conducted quantitative proteomics on these samples.
Outcome:
Pulbication: Hatton, C.F.# Botting, R.A.# Dueñas, M.E.# et. al, Delayed induction of type I and III interferons and nasal epithelial cell permissiveness to SARS-CoV-2, (2021) Nature Communications Dec 7;12(1):7092.
#These authors contributed equally
Two additional manuscripts are underway, one focused on proteomics and another on the MALDI-TOF MS assay.
WP4:
Because of the pandemic and due to travel-restrictions, I was not able to perform my industrial secondment at Boehringer-Ingelheim but will hope to do this in the near future. I did take this opportunity to write a review with my industrial collaborators from GSK and Boehringer-Ingelheim.
Outcome:
Publication: Dueñas, M.E Peltier-Heap, R.E. Leveridge, M., Annan, R.S. Büttner, F.H. Trost, M. Review: Advances in high-throughput mass spectrometry in drug discovery. Submitted to EMBO Molecular Medicine
Outcome: I presented this work (oral talks) at SLAS 2022 (virtually), ACS 2022 (virtually), and ASMS 2022 (in-person). Internally at Newcastle University, I presented this work twice in departmental seminars.
Publication: Marín-Rubio, J.L. Heap, R.E. Dueñas, M.E. Heunis, T., Inns, J., Scott, J., Simpson, A.J. Blair, H., Heidenreich, O., Allan, J.M. Saxty, B. and Trost, M.*, A MALDI-TOF assay identifies nilotinib as an inhibitor of inflammation in acute myeloid leukaemia and multiple myeloma, (2021) biorxiv
WP2:
Once I had the model system validated, I worked on identifying potential fibrotic mediators in primary culture model in a high-throughput manner. I conducted a screening with known inhibitors first to see if that assay was working, followed by library screen using the OpnMe compounds from Boehringer Ingelheim.
WP3:
Unfortunately, we are not in 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. During this time, I developed my expertise in proteomics and conducted quantitative proteomics on these samples.
Outcome:
Pulbication: Hatton, C.F.# Botting, R.A.# Dueñas, M.E.# et. al, Delayed induction of type I and III interferons and nasal epithelial cell permissiveness to SARS-CoV-2, (2021) Nature Communications Dec 7;12(1):7092.
#These authors contributed equally
Two additional manuscripts are underway, one focused on proteomics and another on the MALDI-TOF MS assay.
WP4:
Because of the pandemic and due to travel-restrictions, I was not able to perform my industrial secondment at Boehringer-Ingelheim but will hope to do this in the near future. I did take this opportunity to write a review with my industrial collaborators from GSK and Boehringer-Ingelheim.
Outcome:
Publication: Dueñas, M.E Peltier-Heap, R.E. Leveridge, M., Annan, R.S. Büttner, F.H. Trost, M. Review: Advances in high-throughput mass spectrometry in drug discovery. Submitted to EMBO Molecular Medicine
The Action “MALDI-TOF mass spectrometry metabolite screening assays for drug discovery in human disease” has pushed the frontiers in drug discovery in numerous ways. Current methods for drug discovery in IPF are inadequate. This MALDI-TOF MS assay has the potential to find new drugs for IPF faster, which will benefit the community overall. The assays that I have developed will not only be important for IPF, but also for other diseases. The general applicability is quite wide so it has the potential to be applied to other disease. I am planning to license the MALDI TOF cellular assay to Boehringer-Ingelheim, as well as other pharmaceutical companies.
Moreover, the MSCA has allowed me to develop my leadership and communication skills. During this time, I co-supervised 4 PhD students and was actively engaged in disseminating science to a wider audience (STEMnet Ambassador, Females in Mass spectrometry mentor/mentee program, speaker at Soapbox Science, etc.)
Moreover, the MSCA has allowed me to develop my leadership and communication skills. During this time, I co-supervised 4 PhD students and was actively engaged in disseminating science to a wider audience (STEMnet Ambassador, Females in Mass spectrometry mentor/mentee program, speaker at Soapbox Science, etc.)