The translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is of longstanding medical interest as both a biomarker for neuroinjury and a potential drug target for neuroinflammation and other disorders. Recently it was shown that ligand residence time is a key factor determining steroidogenic efficacy of TSPO-binding compounds. This spurs interest in simulations of (un)binding pathways of TSPO ligands, which could reveal the molecular interactions governing ligand residence time. In this study, we use a weighted ensemble algorithm to determine the unbinding pathway for different poses of PK-11195, a TSPO ligand used in neuroimaging. In contrast with previous studies, our results show that PK-11195 does not dissociate directly into solvent but instead dissociates via the lipid membrane by going between the transmembrane helices. We analyze this path ensemble in detail, constructing descriptors that can facilitate a general understanding of membrane-mediated ligand binding.Correspondence: alexrd @msu.eduThe binding affinity of a ligand to its protein target has long been viewed as the key parameter determining its efficacy. However, recent studies have shown that in some proteinligand systems residence time (RT) correlates more strongly with efficacy than binding affinity (1). Unlike the binding affinity, RT is not a state function; it depends on the height of the free energy barrier separating the bound and unbound states. In order to rationally design ligands for longer RTs we need to understand the (un)binding mechanisma nd what molecular interactions occur along the ligand (un)binding pathway (2-4). Previous studies have shown that the translocator protein 18kDa (TSPO) is one such protein where RT is important for predicting efficacy (5). TSPO is a well-conserved membrane protein, being present all kingdoms including prokaryotes as well as in the outer mitochondrial membrane of eukaryotes (6). TSPO has five transmembrane α-helices (TM1-5) along with a small helical region in a 20-residue loop connecting TM-1 and TM-2 on the cytosolic side ( Fig. 1A). While in the membrane, TSPO is largely found in a dimeric state (7). To date, four different structures have been solved for TSPO structures from both bacterial (7, 8) and mammalian (9, 10) organisms the former by X-Ray crystallography, the latter by NMR. While the structure of TSPO have been solved, its function remains unknown. In humans, TSPO is highly expressed in steroidogenic tissues, leading to the hypothesis that it is involved in the regulation of cholesterol transport across the mitochondrial membrane; indeed, TSPO has been shown to have a high binding affinity for cholesterol (11). There are other studies linking it to apoptosis (12, 13) and cellular stress regulation in TSPO knockout mice (14,15), although evidence for this is mixed (16,17). Increased TSPO expression has also been observed in cases of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases (18). Relatedly, due to its high expression in ar...
