Novel Corrector for Variants of SLC6A8: A Therapeutic Opportunity for Creatine Transporter Deficiency

Abstract

Mutations in creatine transporter SLC6A8 cause creatine transporter deficiency (CTD), which is responsible for 2% of all cases of X-linked intellectual disability. CTD has no current treatments and has a high unmet medical need. Inspired by the transformational therapeutic impact of small molecule "correctors" for the treatment of cystic fibrosis, which bind to mutated versions of the CFTR ion channel to promote its trafficking to the cell surface, we sought to identify small molecules that could stabilize SLC6A8 as a potential treatment for CTD. We leveraged a novel chemoproteomic technology for ligand discovery, reactive affinity probe interaction discovery, to identify small-molecule fragments with photoaffinity handles that bind to SLC6A8 in a cellular environment. We synthesized a library of irreversible covalent analogs of these molecules to characterize in functional assays, which revealed molecules that could promote the trafficking of mutant SLC6A8 variants to the cell surface. Further medicinal chemistry was able to identify reversible drug-like small molecules that both promoted trafficking of the transporter and also rescued creatine uptake. When profiled across the 27 most prevalent SLC6A8 missense variants, we found that 10-20% of patient mutations were amenable to correction by our molecules. These results were verified in an endogenous setting using the CRISPR knock-in of selected missense alleles. We established in vivo proof-of-mechanism for correctors in a novel CTD mouse model with the P544L patient-defined variant knocked in to the SLC6A8 locus, where treatment with our orally bioavailable and brain penetrant tool corrector increased brain creatine levels in heterozygous female mice, validating correctors as a potential therapeutic approach for CTD.

Figure 1

Characterization of SLC6A8 variants by trafficking and transport activity. (A) Schematic of assays developed to identify correctors of SLC6A8 with a trafficking assay to measure surface localization of SLC6A8 and a transport assay to measure creatine transport through SLC6A8 into the cell. (B,C) Trafficking assay measuring surface localized SLC6A8 and intracellular creatine levels detected by RapidFire mass spectrometry of SLC6A8 variants following transient transfection in the PathHunter MEM-EA U-2 OS cells lacking endogenous SLC6A8 (values represent means normalized to wildtype SLC6A8 calculated from n = 2 technical replicates with standard deviation). (D) Correlation between surface localization and residual creatine transport of SLC6A8 variants profiled in (B,C). (E) Residual creatine transport in knock-in variants (values represent mean SLC6A8 knock-in variant as a percentage of wildtype SLC6A8 calculated from n = 3 technical replicates with standard deviation).

Figure 2

Identification of GPA-cooperative and competitive RAPs that label SLC6A8. (A) Chemical structures of creatine and GPA. (B) RAP quantification of labeling SLC6A8 in the presence or absence of GPA. Validated GPA-competitive RAPs are highlighted in blue, and validated GPA-cooperative RAPs are highlighted in red. (C) Chemical structure of RAP 1. (D) SLC6A8 labeling by 20 μM RAP 1 in the presence of indicated concentrations of GPA (values represent mean signal with n = 2 technical replicates with standard deviation). (E) Chemical structure of RAP 2. (F) As described in (D) with cells treated with 20 μM RAP 2 (values represent mean signal with n = 2 technical replicates with standard deviation).

Figure 3

RAPID HTS identified SLC6A8 inhibitors that correct the folding of the mutant transporter. (A) Structure of RAP 3. (B) A library of 50,000 compounds was screened against the RAP displacement probe RAP 3. Hits are highlighted in black with a z score of −3. (C,D) Chemical structures of RAPID HTS hit compound 1 and compound 2, respectively. (E,F) RAP 3 displacement with hits compound 1 and compound 2, respectively. (G,H) Creatine Transport Inhibition Assay: 30 min compound incubation followed by creatine uptake in HEK 293T cells overexpressing wildtype human SLC6A8. (I,J) Trafficking assay: 24 h of compound incubation in U-2 OS cells overexpressing SLC6A8 P544L variant. (K,L) creatine transport correction assay: 24 h compound incubation followed by compound wash off ahead of creatine transport in U-2 OS cells overexpressing SLC6A8 P544L variant. Values represent the mean signal with n = 2 technical replicates with standard deviation.

Figure 4

RAP-derived noncovalent correctors of SLC6A8. (A) Schematic on library derivatization. (B) Chemical structures of compound 3, compound 4, and compound 5. (C) Trafficking of P544L SLC6A8 in the U-2 OS cell line following incubation with compound 3 for 24 h (values represent means normalized to DMSO calculated from n = 2 technical replicates with standard deviation). (D,E) Trafficking and transport assays following treatment of compound 4 for 24 h in the P544L SLC6A8 U-2 OS cell line with compound wash off before creatine transport (values represent means normalized to DMSO calculated from n = 2 technical replicates with standard deviation). (F,G) Trafficking and creatine transport assays following treatment of compound 5 for 24 h in the P544L SLC6A8 U-2 OS cell line with compound wash off before creatine transport (values represent means normalized to DMSO calculated from n = 2 technical replicates with standard deviation).

Figure 5

Increased creatine transport function across SLC6A8 variants (A) Trafficking assay quantifying the surface localization of SLC6A8 variants normalized to wildtype in the presence of DMSO or compound 5 in U-2 OS cells (values represent means normalized to wildtype SLC6A8 from n = 2 technical replicates with standard deviation). (B) Creatine transport correction assay measuring creatine transport of SLC6A8 variants expressed in U-2 OS cells after a 24 h preincubation with either DMSO or compound 5 and wash off of compound ahead of transport (values represent means normalized to wildtype SLC6A8 from n = 2 technical replicates with standard deviation). (C–E) Creatine transport correction assay in the P544L, P554L, and R278C knock-in U-2 OS cell lines following preincubation with compound 5 for 24 h and wash off ahead of transport (values represent means normalized to wildtype SLC6A8 from n = 2 technical replicates with standard deviation).

Figure 6

In vitro and in vivo characterization of compound 6 as a brain-penetrant and bioavailable tool compound. (A) Chemical structure of compound 6. (B) In vitro ADME profile of compound 6. (C) Free drug concentration in plasma and brain homogenate from wildtype C57BL/6J mice following treatment with compound 6 at 30mpk PO at the indicated time points (n = 1 animal per time point). (D) Pulse dose treatment paradigm of compound 6 in vitro to mimic 8 h in vivo brain coverage of EC₅₀ after once daily dosing for 3 days. Creatine transport correction assay was performed in U-2 OS cells with P544L knock-in variant with C14-creatine.

Figure 7

P549L mouse model characterization and compound 6 efficacy. (A) Residual brain creatine for each genotype of SLC6A8 P549L knock-in C57/BL6J mice. Significance was first tested with a Kruskal–Wallis rank sum test (p value of 0.0008) with post hoc analysis using Wilcoxon tests, giving significant p values indicated on the graph (samples were analyzed from three brain regions per animal, n = 3 animals/group). (B) Creatine measured from brain homogenates from heterozygous female mice treated with compound 6 normalized to wildtype female mice treated with vehicle. The heterozygous female mice in the QD group received 4 daily doses of 30 mg/kg, and the heterozygous female mice in the BID group received 2 doses at 0 and 8 h of 30 mg/kg BID. Creatine was measured from three brain regions per mouse (left hemisphere, right cortex, and right hippocampus) and analyzed together for significance tested with a Kruskal–Wallis rank sum test (p value of 0.013) with post hoc analysis using Wilcoxon tests, giving significant p values indicated on the graph (n = 3 animals/group).