Structural Basis for Achieving GSK-3β Inhibition with High Potency, Selectivity, and Brain Exposure for Positron Emission Tomography Imaging and Drug Discovery

Abstract

Using structure-guided design, several cell based assays, and microdosed positron emission tomography (PET) imaging, we identified a series of highly potent, selective, and brain-penetrant oxazole-4-carboxamide-based inhibitors of glycogen synthase kinase-3 (GSK-3). An isotopologue of our first-generation lead, [3H]PF-367, demonstrates selective and specific target engagement in vitro, irrespective of the activation state. We discovered substantial ubiquitous GSK-3-specific radioligand binding in Tg2576 Alzheimer’s disease (AD), suggesting application for these compounds in AD diagnosis and identified [11C]OCM-44 as our lead GSK-3 radiotracer, with optimized brain uptake by PET imaging in nonhuman primates. GSK-3β-isozyme selectivity was assessed to reveal OCM-51, the most potent (IC50 = 0.030 nM) and selective (>10-fold GSK-3β/GSK-3α) GSK-3β inhibitor known to date. Inhibition of CRMP2T514 and tau phosphorylation, as well as favorable therapeutic window against WNT/β-catenin signaling activation, was observed in cells.

High-content image-based analysis and proteomic profiling identifies Tau phosphorylation inhibitors in a human iPSC-derived glutamatergic neuronal model of tauopathy

“…As a positive control for our assay, we used the GSK3 inhibitor CHIR-99021 to induce and test our ability to detect decreases in p-Tau since more than 40 putative phosphorylation sites in tau have been identified as targets of GSK3 61 . This use of CHIR-99021 as a GSK3-inhibiting positive control is further supported by our recent finding that a set of positron emission tomography (PET) ligands that potently inhibit GSK3β in vitro also reduce tau phosphorylation in neurons 51 . In addition to showing that CHIR-99021 reduced tau phosphorylation in our human glutamatergic neurons, we also observe that CHIR-99021 decreased tau levels (Fig.…”

Positron Emission Tomography in Animal Models of Tauopathies

“…No study has so far been reported using SV2A PET in tauopathy animal models. In addition, other emerging targets such as o-GlcNAcase ( Paul et al, 2019 ; Wang et al, 2020 ) and GSK-3β ( Liang et al, 2016 ; Hu et al, 2017 ; Prabhakaran et al, 2017 ; Bernard-Gauthier et al, 2019 ; Varlow et al, 2021 ) that associated with tauopathy remain to be explored in tauopathy models. Pilot study results from microtubule imaging using novel tracer [ 11 C]MPC-6827 in PS19 mice and cynomolgus monkeys showed promising results ( Damuka et al, 2020 ; Sai et al, 2020 ), where a decrease level of [ 11 C]MPC-6827 uptake accompanied by an increased level of tau was detected ( Figure 2E ).…”

Characterization of neuroinflammatory positron emission tomography biomarkers in chronic traumatic encephalopathy

“…Saturation analysis of [ 3 H]PBR-28 and [ 3 H]L-deprenyl in brain homogenates for determination of target affinity ( K d ) and B max was performed with modifications to previously described protocols. 45 , 46 Briefly, frozen aliquots (−80°C) of homogenized (100 mg/ml in Tris-buffered saline, pH 7.4) healthy human brain, or CTE human brain were thawed and diluted 0.25 mg/ml. To define non-specific binding of each [ 3 H]PBR-28 and [ 3 H]L-deprenyl, unlabelled PK-11195 and lazabemide hydrochloride, respectively, [10 mM in dimethyl sulfoxide (DMSO)], were diluted to 100 µM with assay buffer to yield <1% DMSO/buffer.…”