Physiological Consequences of Targeting 14-3-3 and Its Interacting Partners in Neurodegenerative Diseases.

The mammalian 14-3-3 family comprises seven intrinsically unstructured, evolutionarily conserved proteins that bind >200 protein targets, thereby modulating cell-signaling pathways. The presence of 14-3-3 proteins in cerebrospinal fluid provides a sensitive and specific biomarker of neuronal damage associated with Alzheimer's disease (AD), Creutzfeldt−Jakob disease (CJD), spongiform encephalitis, brain cancers, and stroke. We observed significant enrichment of 14-3-3 paralogs G, S, and Z in human brain aggregates diagnostic of AD. We used intra-aggregate crosslinking to identify 14-3-3 interaction partners, all of which were significantly enriched in AD brain aggregates relative to controls. We screened FDA-approved drugs in silico for structures that could target the 14-3-3G/hexokinase interface, an interaction specific to aggregates and AD. C. elegans possesses only two 14-3-3 orthologs, which bind diverse proteins including DAF-16 (a FOXO transcription factor) and SIR-2.1 (a sensor of nutrients and stress), influencing lifespan. Top drug candidates were tested in C. elegans models of neurodegeneration-associated aggregation and in a human neuroblastoma cell-culture model of AD-like amyloidosis. Several drugs opposed aggregation in all models assessed and rescued behavioral deficits in C. elegans AD-like neuropathy models, suggesting that 14-3-3 proteins are instrumental in aggregate accrual and supporting the advancement of drugs targeting 14-3-3 protein complexes with their partners.

Structural modeling of GSK3ß implicates the inactive (DFG-out) conformation as the target bound by TDZD analogs.

Glycogen synthase kinase-3ß (GSK3ß) controls many physiological pathways, and is implicated in many diseases including Alzheimer's and several cancers. GSK3ß-mediated phosphorylation of target residues in microtubule-associated protein tau (MAPTAU) contributes to MAPTAU hyperphosphorylation and subsequent formation of neurofibrillary tangles. Inhibitors of GSK3ß protect against Alzheimer's disease and are therapeutic for several cancers. A thiadiazolidinone drug, TDZD-8, is a non-ATP-competitive inhibitor targeting GSK3ß with demonstrated efficacy against multiple diseases. However, no experimental data or models define the binding mode of TDZD-8 with GSK3ß, which chiefly reflects our lack of an established inactive conformation for this protein. Here, we used metadynamic simulation to predict the three-dimensional structure of the inactive conformation of GSK3ß. Our model predicts that phosphorylation of GSK3ß Serine9 would hasten the DFG-flip to an inactive state. Molecular docking and simulation predict the TDZD-8 binding conformation of GSK3ß to be inactive, and are consistent with biochemical evidence for the TDZD-8-interacting residues of GSK3ß. We also identified the pharmacophore and assessed binding efficacy of second-generation TDZD analogs (TDZD-10 and Tideglusib) that bind GSK3ß as non-ATP-competitive inhibitors. Based on these results, the predicted inactive conformation of GSK3ß can facilitate the identification of novel GSK3ß inhibitors of high potency and specificity.