The solute carriers (SLCs) are a superfamily of membrane transporters with diverse substrates, physiology, and roles in disease. These proteins import and export numerous molecules across the hydrophobic lipid bilayers that define the cell and its internal organelles. These transporters have been associated with numerous pathologies, yet few SLCs have been therapeutically targeted. Underlying the lack of SLC-targeting drugs is their relatively limited characterization. There are numerous reasons for this, often relating to their slow turnover rate and the diversity of substrates that each require a bespoke assay. Furthermore, difficulties in expressing and purifying these targets hamper in vitro studies or the generation of high-affinity renewable binders, such as antibodies or nanobodies.
To resolve this bottleneck and understand SLCs’ physiological role, the RESOLUTE consortium was established to generate open-access tools for these proteins. Funded by the Innovative Medicines Initiative of the European Union and the European Federation of Pharmaceutical Industries and Associations (EFPIA), this public-private partnership worked in two tracks: broadly characterizing the whole family of 450+ SLCs through high-throughput methods, and detailed studies of targets with exceptional biological or medical significance. To identify the latter, a list of priority targets was agreed upon through a discussion among the teams to ensure the best use of additional efforts to generate bespoke assays, binder generation, or detailed biochemical studies.
Among the priority targets, SPNS2 quickly attracted support due to its regulation of lymphocyte migration, role in the pharmacokinetics of the Multiple Sclerosis drug Fingolimod, and potential as a drug target in autoimmune disease. There was broad agreement on the importance of SPNS2 and a recognition that future studies of this protein would benefit from new, open-access tools to study its export of the signaling lipid Sphingosine-1-Phosphate (S1P). Accordingly, several teams with RESOLUTE collaborated to apply their individual group’s expertise in building new resources for SPNS2. Our team in Oxford’s Centre for Medicines Discovery optimized an SPNS2 overexpression and purification method, and handed the resulting protein off to Saša Štefanić at the University of Zurich for nanobody generation. Subsequently, these binders were validated by colleagues from Nuvisan and the Superti-Furga group at CeMM using immunofluorescence, IP-MS, and affinity measurements. In parallel, scientists at Bayer established a high-throughput S1P export assay using an S1PR3-expressing reporter cell line.
Surveying all the available resources created for SPNS2, we realized there was an opportunity to combine this material for a focused study on the transporter’s export of the immunoregulatory S1P and FTY7620-P, and probe the activity of newly reported SPNS2 inhibitors. Further, recent studies by Steel and associates at King’s College London identified mutations of SPNS2 in mice and people, suggesting that S1P is essential in the development of the auditory system. Therefore, we probed the biophysical mechanism for loss of function in these SPNS2 variants that lead to hearing loss. This required significant work and close coordination of each team, including CryoEM structures with the nanobody to probe S1P binding, making stable cell lines for scientific or clinical SPNS2 mutants to be investigated in the S1P export assay, and adapting the immunofluorescence platform to characterize the trafficking of SPNS2 variants. Finally, demonstrating the collaborative and open nature of the RESOLUTE program, colleagues from the Khalid group in Oxford’s Department of Biochemistry joined the study to provide key molecular dynamics results that revealed the inherent mobility of substrate within the SPNS2 central cavity and subtle differences in binding for substrates and inhibitors.
Our results, in conjunction with studies by Chen et al., Duan et al., and Pang et al., provide a near-complete description of SPNS2’s reaction cycle in exporting S1P and FTY720-P. Satisfying, each team approached the challenges of solving SPNS2’s structure or measuring its transport activity using distinct methods, yet generally came to similar conclusions for the protein’s substrate binding and enzymatic mechanism.
Collectively, the RESOLUTE resources provide the field with a rich toolbox for probing the protein’s activity in normal or aberrant physiology, or screening and characterizing new SPNS2-targeting therapeutics. As a highlight of these resources’ value, in a second collaboration on SPNS2, we worked with the Robinson group at Oxford to uncover the second messenger PIP2 as an unexpected regulation of the protein’s S1P export. These results dovetailed nicely with the SPNS2 structure, as the disordered N-terminus in our CryoEM maps is explained by the necessary mobility of this region to retrieve PIP2 from the inner leaflet for subsequent regulation of the transporter’s central MFS transport domain.
This SPNS2 work and other flagship RESOLUTE studies have become an excellent showcase for the effective collaboration between academic and pharma partners to generate high-quality tools and data for studying SLCs. In keeping with the open-access philosophy of the project, detailed protocols are available on a Zenodo repository (https://zenodo.org/communities/resolute), scientific outputs are available on the RESOLUTE website (https://re-solute.eu/), and clones can be freely requested from Addgene.