The translocator protein (TSPO) (18 kDa) is an emerging drug target for the treatment of numerous
pathologies including cancer and neurodegenerative disease. However, our limited knowledge of TSPO
binding site(s) has hindered the development of TSPO ligands with potential therapeutic effects. We have
synthesized a series of pyrrolobenzoxazepines (1-10) to better characterize the interaction of ligands with the
TSPO across species, and to determine their functional profiles. All ligands 1-10 displaced the binding of
[3H]PK 11195 to the TSPO at nanomolar concentrations, with discrepancies in binding affinity between rat and
human TSPO. Interestingly, non-linear regression analysis revealed that some ligands bound to the protein
with a Hill slope not equal to 1.0, suggesting possible additional TSPO binding sites with allosteric effects.
However, this trend was not conserved between rat and human. When tested for their effects on pregnenolone
production in rat C6 glioma cells, nitric oxide release in murine microglia, and cell proliferation in human MCF-7
breast cancer cells, the pyrrolobenzoxazepines (40 μM) displayed functional effects which did not correlate to
the binding trend observed in competition assays. We propose that consideration of species differences and
binding site cooperativity, plus optimization of currently accepted functional assays, will aid in the development
of drugs targeting TSPO that can be used as therapeutics for human disease.