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Genomic instability caused by a deficiency in the DNA damage response and repair has been linked to age-related cognitive decline and neurodegenerative diseases. Preventing genomic instability that ultimately leads to neuronal death may provide a broadly effective strategy to protect against multiple potential genotoxic stressors. Recently, the zinc-dependent, class I histone deacetylase HDAC1 has been identified as a critical factor for protecting neurons from deleterious effects of DNA damage in Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Translating these observations to a novel neuroprotective therapy for AD, ALS, and FTD may be advanced by the identification of small molecules capable of increasing the deacetylase activity of HDAC1 selectively over other structurally similar HDACs. Here, we demonstrate that exifone, a drug previously shown to be effective in treating cognitive deficits associated with AD and Parkinson’s disease, the molecular mechanism of which has remained poorly understood, potently activates the deacetylase activity of HDAC1 and provides protection against genotoxic stress. We show that exifone acts as a mixed, non-essential activator of HDAC1 that is capable of binding to both free and substrate-bound enzyme resulting in an increased relative maximal rate of HDAC1-catalyzed deacetylation. Exifone can directly bind to HDAC1 based upon biolayer interferometry assays with kinetic and selectivity profiling suggesting HDAC1 is preferentially targeted compared to other class I HDACs and the kinase CDK5 that have also been implicated in neurodegeneration. Consistent with a mechanism of deacetylase activation intracellularly, treatment of human induced pluripotent stem cell (iPSC)-derived neuronal cells resulted in globally decreased histone acetylation. Moreover, exifone treatment was neuroprotective in a tauopathy patient iPSC-derived neuronal model subject to oxidative stress. Taken together, these findings reveal exifone as a potent activator of HDAC1-mediated deacetylation, thereby offering a lead for novel therapeutic development aiming to protect genomic integrity in the context of neurodegeneration and aging.Graphical 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, [(3)H]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 [(11)C]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 (IC(50) = 0.030 nM) and selective (>10-fold GSK-3β/GSK-3α) GSK-3β inhibitor known to date. Inhibition of CRMP2(T514) and tau phosphorylation, as well as favorable therapeutic window against WNT/β-catenin signaling activation, was observed in cells.

An aldol-based ‘build/couple/pair’ (B/C/P) strategy was applied to generate a collection of stereochemically and skeletally diverse small molecules. In the build phase, a series of asymmetric syn- and anti- aldol reactions were performed to produce four stereoisomers of a Boc protected γ-amino acid. In addition both stereoisomers of O-PMB-protected alaninol were generated to provide a chiral amine coupling partner. In the couple step, eight stereoisomeric amides were synthesized by coupling the chiral acid and amine building blocks. The amides were subsequently reduced to generate the corresponding secondary amines. In the pair phase, three different reactions were employed to enable intramolecular ring-forming processes, namely: nucleophilic aromatic substitution (SNAr), Huisgen [3+2] cycloaddition and ring-closing metathesis (RCM). Despite some stereochemical dependencies, the ring-forming reactions were optimized to proceed with good to excellent yields providing a variety of skeletons ranging in size from 8- to 14-membered rings. Scaffolds resulting from the RCM pairing reaction were diversified on solid-phase to yield a 14,400-membered library of macrolactams. Screening of this library led to the discovery of a novel class of histone deacetylase inhibitors, which display mixed enzyme inhibition and led to increased levels of acetylation in a primary mouse neuron culture. The development of stereo-structure/activity relationships (SSAR) was made possible by screening all 16 stereoisomers of the macrolactams produced through the aldol-based B/C/P strategy.

Carboxylic acids with known central nervous system and histone deacetylase (HDAC) inhibitory activities were converted to hydroxamic acids and tested using a suite of in vitro biochemical assays with recombinant HDAC isoforms, cell based assays in human cervical carcinoma HeLa cells and primary cultures from mouse forebrain, and a whole animal (Xenopus laevis) developmental assay. Relative to the parent carboxylic acids, two of these analogues exhibited enhanced potency, and one analogue showed altered HDAC isoform selectivity and in vivo activity in the Xenopus assay. We discuss potential uses of these novel hydroxamic acids in studies aimed at determining the utility of HDAC inhibitors as memory enhancers and mood stabilizers.

Mammalian and yeast cells contain three isozymes of isocitrate dehydrogenase: mitochondrial NAD- and NADP-specific enzymes and a cytosolic NADP-specific enzyme. Independent metabolic functions of these enzymes in Saccharomyces cerevisiae were examined by analyses of expression and of phenotypes displayed by mutants containing all possible combinations of isozyme gene disruptions. All three isocitrate dehydrogenases are expressed at high levels with growth on nonfermentable carbon sources, whereas the mitochondrial NADP-specific enzyme constitutes the major cellular activity with growth on glucose. Distinct growth phenotypes are observed for mutants expressing a single isozyme, and expression of at least one isozyme is necessary for glutamate-independent growth. The NADP-specific tricarboxylic acid cycle isocitrate dehydrogenase from Escherichia coli was expressed in mitochondrial and cytosolic compartments of the yeast disruption mutants using plasmids carrying gene fusions of yeast promoters and a mitochondrial targeting presequence with the bacterial coding sequence. The bacterial enzyme is competent for restoration of NADP-specific functions in either compartment but does not compensate for function of the yeast NAD-specific tricarboxylic acid cycle enzyme.