title: Development of an Automated MALDI Mass Spectrometry Assay for direct Analysis of Cellular Drug Uptake via the Organic Anion Transporting Polypeptide OATP2B1
creator: Unger, Melissa Simone
subject: ddc-500
subject: 500 Natural sciences and mathematics
subject: ddc-610
subject: 610 Medical sciences Medicine
description: For the longest time, there was the widely held belief that drug uptake into a cell is mainly due to diffusion, channels and carriers. Only in the 1940s, there was the first drug-transporter interaction discovered. As more transporter-related diseases where discovered and transport proteins identified that had an important connection to cancer, like the breast cancer resistance protein, the research field gained more interest. Drug-Drug interactions were identified and an International Transporter Consortium was founded that had the task to identify relevant transport proteins and closely work together with the drug approval by the FDA. The majority of transport proteins either belong to the class of ABC transporters, which are primary active export transporters or to the class of solute carrier (SLC) transporters, which are secondary active uptake transporters. OATP2B1 is an organic anion transporting polypeptide and is part of the group of SLC transporters. It is seen as an emerging transport protein and has gained attention due to many drug-fruit juice interactions. As the simultaneous intake of fruit juices and drugs are likely to happen in everyday life and OATP2B1 is a human transporter expressed mostly in liver and intestine, it is important to understand more about its uptake mechanism and possible inhibition.    Until now, the emerging field of transporters are examined by either radioactive or fluorescence-based assays. Radioactive assays render a rather unpopular method with many obstacles like cost, safety-issues, labelling and no possibility of high-throughput. Fluorescence-based assays are widespread and have the positive property that they can be automated and used in HTS. The negative aspects here are also labelling and the false negatives and positives prediction that comes through autofluorescence and quenching effects. Our goal therefore was to develop an alternative cell based assay based on a different technology: MALDI MS. MALDI MS brings the advantage that it is a label-free technique and it also is HTS-compatible, like it was already shown in the past. Cell-based MALDI MS assays have gained recognition in the last years in consequence of their speed, robustness and ease in setup and are suitable for the investigation of transport mechanisms due to the abundance of additional information gathered by this technique.    For the MALDI MS method development, the use of E3S as a substrate provided the best results. The optimal matrix composition for the detection of E3S in the cells had to be found. 2.5 mg/mL Ph-CCA-NH2 in 70 % ACN were identified as the best composition and were further used for transport characterisation with the confirmation of time-dependence and concentration-dependence of E3S uptake. The optimal assay conditions (2 min, 10 µM E3S) were used to screen a set of 294 compounds consisting of the top 300 marketed drugs and a set of compounds that are known to interact with OATP2B1. The used compounds were tested in their ability to inhibit the uptake of E3S into the cells. There were 76 compounds found with an inhibition of more than 50 %, which were then further analysed by pIC50 determination. 67 of those compounds could be verified as hits, leading also to 14 very potent inhibitors with a pIC50 over 6. With an average CV % under 10 for 6 biological replicates, the method confirms reproducibility and reliability of the data.     As a reference assay to the aspired MALDI MS assay, also a fluorescence-based assay was developed to examine the uptake of DBF through OATP2B1. This assay was also used to screen the 294 compound set and 67 compounds with an inhibition ≥50 % were identified in the course of the experiments. 57 could be verified as a hit. There was a calculation being done to examine the clinical relevance of those transporters showing a clinical relevance of more than 60 % in the intestine reinforcing the meaning of transporter studies. By the comparison of the two techniques, it was found that only 47 inhibitors overlapped leading to compounds that were not found with one of both methods. This can eventually be explained by the use of different substrates and the multiple binding sites of OATP2B1, but still has to be addressed further. The comparison of the data with the previously published Karlgren et al. data set showed an overlap of 83 % and therefore shows the applicability of the MALDI MS method. To conclude, there were two assay systems developed that are suitable to examine the emerging transport protein OATP2B1. The importance of this transport protein has been shown through the amount of identified (clinically relevant) inhibitors. While the developed fluorescence-based method acts as a good reference method, the newly developed MALDI MS method represents a completely new way to analyse substrate and inhibitor of transporters. With its ease and speed in handling and most notably the label-free approach, the MALDI MS method is an indispensable tool for transporter characterisation and DDI analysis.
date: 2021
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/29343/1/Dissertation_MU.pdf
identifier: DOI:10.11588/heidok.00029343
identifier: urn:nbn:de:bsz:16-heidok-293436
identifier:   Unger, Melissa Simone  (2021) Development of an Automated MALDI Mass Spectrometry Assay for direct Analysis of Cellular Drug Uptake via the Organic Anion Transporting Polypeptide OATP2B1.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/29343/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng