Unraveling 4-Phenylbutyrate's Therapeutic Role in SLC6A1 Disorders: Pharmacochaperoning Over HDAC Inhibition

Variants in SLC6A1, encoding the GABA transporter 1 (GAT-1), cause epilepsy, autism spectrum disorder, and developmental delay via loss of GABA uptake, impaired trafficking, and ER retention. We previously found that 4-Phenylbutyrate (PBA), an FDA-approved drug, restores GABA uptake and reduces seizures in SLC6A1-related disorders, prompting a phase I clinical trial (NCT04937062) primarily designed to evaluate safety and tolerability of Ravicti (PBA). However, its exact mechanism, pharmacochaperoning or histone deacetylase (HDAC) inhibition, remains unclear. This study compares PBA with pharmacochaperones and HDAC inhibitors to determine how it restores GAT-1 function in the cell and mouse models.

We evaluated the function for 32 SLC6A1 variants and two representative mouse models at baseline and various treatment options using ³H GABA uptake assays. We evaluated the effect of PBA in comparison with other chaperone inducers or HDAC inhibitors on the mutant and the wild-type GAT-1 expression. Importantly, we evaluated the effect of chaperone inducers and HDAC inhibitors on GAT-1 function and seizures in two representative knock-in mouse models, Slc6a1^+/A288V and Slc6a1^+/S295L, with EEG recordings.

PBA restored GABA uptake and GAT-1 surface expression across all variants, and TUDCA mimicked the effects of PBA. HDAC inhibitors exhibited modest rescue in vitro but failed to restore GAT-1 function or mitigate seizures in the knockin mice.

PBA acts as a pharmacochaperone, not an HDAC inhibitor, to restore GAT-1 function and reduce seizure burden in the diseased mice, supporting pharmacochaperoning as the major mechanism for rescuing SLC6A1-related disorders.