Novel Corrector\nfor Variants of SLC6A8: A Therapeutic\nOpportunity for Creatine Transporter Deficiency

Mutations in creatine\ntransporter SLC6A8 cause creatine\ntransporter\ndeficiency (CTD), which is responsible for 2% of all cases of X-linked\nintellectual disability. CTD has no current treatments and has a high\nunmet medical need. Inspired by the transformational therapeutic impact\nof small molecule “correctors” for the treatment of\ncystic fibrosis, which bind to mutated versions of the CFTR ion channel\nto promote its trafficking to the cell surface, we sought to identify\nsmall molecules that could stabilize SLC6A8 as a potential treatment\nfor CTD. We leveraged a novel chemoproteomic technology for ligand\ndiscovery, reactive affinity probe interaction discovery, to identify\nsmall-molecule fragments with photoaffinity handles that bind to SLC6A8\nin a cellular environment. We synthesized a library of irreversible\ncovalent analogs of these molecules to characterize in functional\nassays, which revealed molecules that could promote the trafficking\nof mutant SLC6A8 variants to the cell surface. Further medicinal chemistry\nwas able to identify reversible drug-like small molecules that both\npromoted trafficking of the transporter and also rescued creatine\nuptake. When profiled across the 27 most prevalent SLC6A8 missense\nvariants, we found that 10–20% of patient mutations were amenable\nto correction by our molecules. These results were verified in an\nendogenous setting using the CRISPR knock-in of selected missense\nalleles. We established in vivo proof-of-mechanism for correctors\nin a novel CTD mouse model with the P544L patient-defined variant\nknocked in to the SLC6A8 locus, where treatment with our orally bioavailable\nand brain penetrant tool corrector increased brain creatine levels\nin heterozygous female mice, validating correctors as a potential\ntherapeutic approach for CTD.