A considerable amount of ground-work had to be established, thought to be necessary to enable GameofGates. This had to do both with establishing the biological parameters as well as with the development of the technical tools necessary. In terms of biological parameters we thought to explore the level of expression plasticity of SLCs in response to changes in environment and metabolic conditions. In the initial year, a large proportion of energy was spent trying to learn how to change growth culture conditions without completely upsetting cellular growth. This turned out to be a difficult thing to do. Ultimately, we opted to remove one nutrient at the time and perform gain of function screens. This worked out very well for amino acids and eventually we found, via a genetic approach that the cellular milieu influences the cellular SLC profile. The most important work has been enabled by the creation of a SLC-focused CRISPR library that due to its reduced size compared to genome-wide, allowed for more screens with high statistical depth. The work over the period has produced evidence for a role of SLCs in cell fitness (“essentiality”), immune function, cell death, epigenetic regulation, pH homeostasis and drug transport. Moreover, we have been working on some new approaches to test engagement of chemical matter, drugs and metabolites, to SLC transporters. This has been achieved through the monitoring of thermal stabilization of proteins in cells. Though stabilization could also be the result of indirect effects, in the majority of cases, direct binding and alteration of thermodynamic properties through direct binding is likely to be the cause. Additional breakthrough in terms of technological approach to SLC function comes from experimental strategies we developed that allow screening for drug-gene and gene-gene relationships effectively. Both technologies have empowered studies that resulted in findings that allow to conclude that many drugs need (at least one) SLC transporters to unfold their action and via genetic interaction profiles we could identify new SLCs. On one hand, these results will allow us to create new assays that are functionally dependent on individual SLCs and thus exploitable for drug discovery and on the other hand they have provided the basis for an IMI 2 grant (RESOLUTE;
https://re-solute.eu/) dedicated to scale up and broaden these approaches to more cell types. Our laboratory already has published more than 20 papers on transporters, partly sponsored by the GameofGates grant.