Choline is an essential nutrient that underpins multiple metabolic pathways. It is the precursor of phosphatidylcholine, the most abundant membrane phospholipid, which is metabolised to betaine, a methyl donor for DNA, RNA, and protein methylation, and converted to acetylcholine in cholinergic neurons – the neurotransmitter essential for learning and memory1. Activated choline metabolism is a hallmark of malignant cancers, including glioblastoma multiforme – the most aggressive form of primary brain cancer2. Tumour cells exhibit an elevated demand for choline due to its critical role in supporting cellular proliferation and malignant transformation. This link is so strong that choline positron emission tomography scans are commonly used in the clinic to diagnose cancer and monitor tumour growth3.
Recently, our lab discovered that FLVCR2 is the major choline transporter at the blood-brain barrier and revealed structural and mechanistic details of this process4. Interestingly, FLVCR2 expression is significantly increased in both low-grade glioma and glioblastoma multiforme, and elevated FLVCR2 expression is linked to worse survival rates in glioma patients5.
Our lab has been working on developing inhibitors of FLVCR2 as a potential novel therapy for glioblastoma multiforme. Although FLVCR2 inhibition has shown promise, current inhibitors also affect its close homologue FLVCR1, which shares 55% sequence identity and is broadly expressed in peripheral tissues. Such off-target inhibition presents a major obstacle for developing FLVCR2-selective therapeutics. While structures of FLVCR2 in both the outward- and inward-facing state have been resolved, the outward-facing state of FLVCR1 remains unsolved, limiting our ability to develop FLVCR2-specific inhibitors.
To overcome this limitation, we aim to determine the cryo-EM structures of FLVCR1 in the outward-facing state in the absence of substrate, and in the presence of its two substrates: choline and ethanolamine. Thus far, we have successfully expressed and purified human FLVCR1, and confirmed complex formation with a Fab that stabilises the outward-facing state. These structures will provide mechanistic insight into FLVCR1-mediated transport and enable rational design of FLVCR2-selective inhibitors with reduced off-target activity. Together, this work will expand our understanding of choline transport, and support the development of targeted therapies for glioblastoma mutiforme.