Elucidating hepatic OATP-ligand interactions and selectivity

The human hepatic organic anion transporting polypetides (OATPs), termed OATP1B1, OATP1B3, and OATP2B1 are important for proper liver function because they mediate the uptake of a variety of substances into the liver. However, it is not known properly how the three different transporters work, their communalities and differences. Therefore, we want to understand how different molecules or drugs interact with the three different proteins on a molecular basis in order to learn about the driving forces for the binding and selectivity (preferred binding to one of the three). In the next step, this knowledge will be used to identify new molecules acting on one or several of the hepatic OATPs. What are our Hypotheses? The three hepatic OATPs do possess communalities and differences regarding their protein structure. Those structural differences are influencing the way in which different molecules/drugs are recognized by the transporters, and thus their biological effects on the transporters might be different. Elucidating hepatic OATP-ligand interaction will therefore give insights into the molecular basis of how these transporters work and shed light on reasons for compound selectivity. Which Methods do we use? This project is designed as an interdisciplinary research approach, combining computational modelling of protein structures and compound-protein interactions with pharmacological testing of compound activity. Computer-generated structural models for the three hepatic OATPs (OATP1B1, OATP1B3, and OATP2B1) will be constructed on basis of other protein structures with a high degree of shape similarity. Interactions of the three hepatic OATPs with molecules/drugs will be identified by applying `ligand-protein docking` of a large number of molecules, a method which computationally predicts the preferred orientation of a molecule when placed into a protein. Novel compounds potentially targeting OATPs will be predicted on basis of the computer models and experimentally tested for their activity. What is new and special about this project? Up to now, only very few computer-generated structural models are available for hepatic OATPs and no systematic large-scale docking study has been performed so far. In addition, our study for the first time compares the molecule-protein interactions between all three hepatic OATPs, and thus will give insights in reasons for compound selectivity. Moreover, our study will identify new hepatic OATP ligands which will serve for further functional characterization of the proteins. Moreover, knowledge about new drug-OATP interactions will help to increase the safety of medication schemes in clinics, because if one drug is blocking the transporter the other drug cannot be taken up into the liver anymore. This frequently causes severe side effects during drug therapy, which might be prevented by the findings of our study.

ELUCIDATING HEPATIC OATP-LIGAND INTERACTIONS AND SELECTIVITY Background & Aims: The human hepatic organic anion transporting polypeptides (OATPs) - termed OATP1B1, OATP1B3, and OATP2B1 - are transport proteins important for proper liver function because they mediate the uptake of a variety of substances (ligands) into the liver. Interaction of some drugs with these proteins can lead to unwanted adverse effects, termed drug-drug interactions, when substances are administered simultaneously. When starting this project, it was not properly understood how the three transporters interact with different ligands. Therefore, the main goal of this project was to elucidate the molecular basis for ligand interaction and selectivity of these transporters. Gathering this knowledge, can aid to prevent clinically important drug-drug interactions. This could be achieved by the creation of computer-based models for these proteins and simulation of the binding process of molecules (by a technique called molecular docking). Further, the models have been used to identify new molecules interacting with one or several of these transporters. To validate the computer predictions, biochemical methods have been used to assess the binding affinity of the novel molecules. What did we find out? In this project we identified particular binding regions in the studied transporters which seem to be important for the recognition of interacting molecules. We could see differences in interaction patterns comparing the three transporters, while differences are more pronounced when comparing OATP1B1 or OATP1B3 to OATP2B1. We could also pinpoint specific amino acid residues that might cause selectivity of a molecule for a specific transporter. Furthermore, by investigating the dynamic movements of a group of structurally similar proteins, we could identify regions that fluctuate more, potentially being more involved in the recognition of molecules or function of the protein. Finally, when using our structural models for the purpose of detecting new interacting ligands (by a technique called virtual screening) we identified six new medium to strong affinity inhibitors for the various transporters. Which challenges did we face? When starting this project, bioactivity data for OATP2B1 was still quite limited and no comparative study for all three proteins has been done before. Thus, an extensive data collection effort had to be undertaken. Moreover, transport proteins are difficult to model computationally due to a lack of structural templates. We therefore decided to use a technique which compares protein structures across protein families. Furthermore, transporters aren't static proteins so that it was required to include protein flexibility into the modeling procedure. What are the implications? The newly identified ligands can be used for further functional studies on these transporters. The knowledge gathered about the transporters, their commonalities and differences help to avoid unwanted drug-drug interactions. The developed computational modeling workflows can be re-used to model other (challenging) targets.