Discovery of high affinity γ‐Hydroxybutyrate (GHB) Receptor Ligands for SSADH Deficiency, a Disorder of GABA Metabolism
Authors: Chen Justin , Szlenk Christopher T. , McConnell Alice , Roullet Jean-Baptiste O. , Gibson K. Michael , Natesan Senthil 
Published: April 2020

ObjectiveSuccinic semialdehyde dehydrogenase deficiency (SSADHD) is the most prevalent inherited disorder of γ‐aminobutyric acid (GABA) metabolism. SSADHD is caused by mutations in the ALDH5A1 gene resulting in impaired enzyme activity. Functional deficiency of SSADH prevents conversion of succinic semialdehyde to succinic acid, resulting in excessive accumulation of neurotransmitters GABA and its analog γ‐hydroxybutyrate (GHB). The disease presents with non‐specific mild to moderate developmental delay, severe expressive language impairment, epilepsy, and neuropsychiatric problems. Blocking GHB receptors (GHBR) with the specific antagonist NCS‐382 has been shown to rescue SSADH‐deficient mice from premature lethality suggesting antagonism of GHB effects may be therapeutically beneficial to patients. Due to a lack of structural information for the GHBR, a virtual screening strategy using a ligand‐based pharmacophore model, followed by in vitro [3H]NCS‐382 binding displacement assay, was used to identify high‐affinity small molecules as potential GHBR antagonists. Here, we report the potential binding site map of GHBR and discovery of novel and structurally diverse high‐affinity GHBR ligands as potential lead compounds for further development as possible therapeutics in SSADHD.MethodsA feature‐ and geometry‐based pharmacophore model was developed in MOE software using a set of ~110 structurally diverse compounds with known experimental binding affinities for GHBR. The iteratively refined model exhibited high accuracy in distinguishing active compounds from inactive ones and was further validated with an external test set of 25 compounds with known GHBR affinities. The validated model was used to screen commercially available CNS‐focused libraries containing several hundreds of thousands of compounds. The top hit molecules with desirable physico‐chemical and pharmacokinetic properties were chosen for the [3H]NCS‐382 binding displacement assay. The in vitro assay results were used to establish structure‐activity relationships to guide further optimization of the pharmacophore model, whose refined version was used for subsequent screening.ResultsWe have obtained the potential binding site map of GHBR by developing and refining a ligand‐based pharmacophore model iteratively based on in vitro assay results. The refined pharmacophore model provides insights into structural features that are essential for binding to the GHB receptor. After three rounds of combined in silico and in vitro assessments, we discovered several structurally diverse lead compounds exhibiting low μM affinity to the GHB receptor.ConclusionsThis study represents a strategic application of ligand‐based pharmacophore model‐based virtual screening and in vitro [3H]NCS‐382 binding displacement assay to identify high affinity ligands for the putative GHB receptor. The obtained lead compounds are being subjected to further refinement of their GHBR binding activity prior to preclinical testing of their pharmacodynamic and pharmacokinetic profile.Support or Funding InformationNational Institutes of Health ‐ NINDS R41NS107099‐01Pharmacophore‐based virtual screening for GHBR ligandsFigure 1