High Throughput Screen to Identify Glutamate Carboxypeptidase II (GCPII) Brain-Penetrable-Inhibitors for the Treatment of Pain

National Center For Advancing Translational Sciences

Investigators

Abstract

Glutamate carboxypeptidase II (GCPII) is a membrane-bound, zinc metalloenzyme that catalyzes the hydrolysis of the abundant neuropeptide N-acetyl-aspartate-glutamate (NAAG) to N-acetyl-aspartate (NAA) and glutamate. Inhibition of GCPII in the brain results in both increased extracellular NAAG and decreased extracellular glutamate. These effects diminish glutamate transmission, which has been associated with pain sensation and transmission. Previous studies indicated that GCPII inhibition would be a viable approach to treating pain; however, the GCPII inhibitors evaluated to date have not moved into the clinic because of poor brain penetration, low oral bioavailability, and low membrane permeability. These limitations will be addressed by the collaborative team as they focus on the identification of small molecule inhibitors of GCPII that penetrate the brain for the treatment of pain. Previously, the team developed a 384-well GCPII Dual Stream Liquid Chromatography with Tandem Mass Spectrometry (LC/MS/MS) assay for this project. It led to the establishment of an MS-based HTS group at NCATS. To date, the team screened over 30,000 compounds and hundreds of thousands more virtually. Promising hits were shipped to the collaborator and validated in their radioactivity-based assay. Unfortunately, many hits failed to confirm with fresh powders, leading to expanded and follow-up screening. This year, the team developed two biophysical assays to complement the enzymatic assay workflow. These assays will provide hit validation through binding and reduce unnecessary resynthesis and validation of false positives. The high-throughput workflow also enables validation and affinity ranking of hundreds of hits per run. Results from the initial nanoDSF assay pilot testing are promising, and HTS hit testing is ongoing. The team has also developed an SPR assay that they are pilot testing with prior art to further validate prior HTS hits. This work has identified several novel chemotypes that are currently being validated and profiled. Additionally, these assays will be employed to validate DEL hits obtained independently by Dr. Slusher. The team has already published one paper (PMID: 38867136), and a second paper about the discovery of novel GCPII inhibitor scaffolds is under review. A third manuscript is in progress