Elucidation of the GSK3α Structure Informs the Design of Novel, Paralog-Selective Inhibitors.

Glycogen synthase kinase 3 (GSK3) remains a therapeutic target of interest for diverse clinical indications. However, one hurdle in the development of small molecule GSK3 inhibitors has been safety concerns related to pan-inhibition of both GSK3 paralogs, leading to activation of the Wnt/ß-catenin pathway and potential for aberrant cell proliferation. Development of GSK3α or GSK3ß paralog-selective inhibitors that could offer an improved safety profile has been reported but further advancement has been hampered by the lack of structural information for GSK3α. Here we report for the first time the crystal structure for GSK3α, both in apo form and bound to a paralog-selective inhibitor. Taking advantage of this new structural information, we describe the design and in vitro testing of novel compounds with up to ∼37-fold selectivity for GSK3α over GSK3ß with favorable drug-like properties. Furthermore, using chemoproteomics, we confirm that acute inhibition of GSK3α can lower tau phosphorylation at disease-relevant sites in vivo, with a high degree of selectivity over GSK3ß and other kinases. Altogether, our studies advance prior efforts to develop GSK3 inhibitors by describing GSK3α structure and novel GSK3α inhibitors with improved selectivity, potency, and activity in disease-relevant systems.

Discovery of Phospholipase D Inhibitors with Improved Drug-like Properties and Central Nervous System Penetrance.

Phospholipase D (PLD) is a phospholipase enzyme responsible for hydrolyzing phosphatidylcholine into the lipid signaling molecule, phosphatidic acid, and choline. From a therapeutic perspective, PLD has been implicated in human cancer progression as well as a target for neurodegenerative diseases, including Alzheimer's. Moreover, knockdown of PLD rescues the ALS phenotype in multiple Drosophila models of ALS (amyotrophic lateral sclerosis) and displays modest motor benefits in an SOD1 ALS mouse model. To further validate whether inhibiting PLD is beneficial for the treatment of ALS, a brain penetrant small molecule inhibitor with suitable PK properties to test in an ALS animal model is needed. Using a combination of ligand-based drug discovery and structure-based design, a dual PLD1/PLD2 inhibitor was discovered that is single digit nanomolar in the Calu-1 cell assay and has suitable PK properties for in vivo studies. To capture the in vivo measurement of PLD inhibition, a transphosphatidylation pharmacodynamic LC-MS assay was developed, in which a dual PLD1/PLD2 inhibitor was found to reduce PLD activity by 15-20-fold.