Automatable microplate-based in vitro assays for screening intestinal drug transport and metabolism
Year: 2012
Type: dissertation
Abstract: Automation compatibility is a prerequisite for in vitro transport and metabolism assays that are designed for screening large numbers of compounds at the early stages of drug discovery, in parallel with activity optimization. However, automation of more complex assays has also many benefits, even if it does not always increase the throughput of the assay directly. In this thesis work, a new automatable high throughput screening assay was developed for detecting different compound interactions with UGT1A6, one of the human intestinal UGT enzymes. The fluorescence-based assay relies on a robust probe reaction, glucuronidation of 1 naphthol by recombinant human UGT1A6, which yields a highly fluorescent product, 1 naphthylglucuronide. Under the optimized assay conditions, the plate reader-based analysis method was able to detect a given compound interaction with the enzyme, through the probe reaction, in a comparable manner to the (cumbersome) reference HPLC-based method. The new method can analyze the interaction of many different compounds with the UGT1A6 simultaneously. The developed assay was then used to collect data for computational model development. The effects of different compounds on the probe glucuronidation rate were used for a classification model, based on an SVM method, and for 3D-QSAR models, based on CoMFA and CoMSIA analyses. Both the models indicated physicochemical parameters and functional groups that are important for the interaction of different compounds with the UGT1A6. Interactions of two drugs, diclofenac and indomethacin, were studied more thoroughly with the several UGT enzymes, including UGT1A6. The effect of pH on the interaction was examined in both human liver and intestinal microsomes, as well as with many individual recombinant human UGTs. The results indicated that the effects of pH were both enzyme and substrate dependent, suggesting that changes in the enzyme, probably protonation of one or more amino acid side chain, played a major role in the observed effects. Finally, automation of three different types of cell-based assays, Caspase-Glo® 3/7, sulforhodamine B and bidirectional Caco-2 monolayer transport assays, are described in detail. Although, the bidirectional Caco-2 assay was the most challenging for automation, due to the complex assay protocol and the sensitivity of the monolayer to mechanical stress, mainly caused by pipetting, automation improved the utilization potential of the laborious method significantly.
Source: Työväentutkimus Vuosikirja
Author: Anne Soikkeli
Language: English
Subfield: Pharmacology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2132792179