The molecular determinants for the activities of the reported benzoic acid (SH4-54), salicylic acid (BP-1-102), and benzohydroxamic acid (SH5-07)-based STAT3 inhibitors were investigated to design optimized analogues. All three leads are based on an -methylglycinamide scaffold, with its two amine groups condensed with three different functionalities. The three functionalities and the CH group of the glycinamide scaffold were separately modified. The replacement of the pentafluorobenzene or cyclohexylbenzene, or replacing the benzene ring of the aromatic carboxylic or hydroxamic acid motif with heterocyclic components (containing nitrogen and oxygen elements) all decreased potency. Notably, the Ala-linker analogues, and, and the Pro-based derivative , all with ()-configuration at the chiral center, had improved inhibitory activity and selectivity against STAT3 DNA-binding activity , with IC of 3.0 ± 0.9, 1.80 ± 0.94, and 2.4 ± 0.2 μM, respectively. Compounds ,, , and other analogues inhibited constitutive STAT3 phosphorylation and activation in human breast cancer and melanoma lines, and blocked tumor cell viability, growth, colony formation, and migration. Pro-based analogue, , with a relatively polar tetrahydropyranyl (THP) ring, instead of the cyclohexyl, showed improved permeability. In general, the ()-configuration Pro-based analogs showed the overall best profile, including physicochemical properties (e.g., microsomal metabolic stability, Caco-2 permeability), and in particular, showed improved tumor-cell specificity.

Understanding the specific gene changes underlying the prodromic stages of Alzheimer's disease pathogenesis will aid the development of new, targeted therapeutic strategies for this neurodegenerative disorder. Here, we employed RNA-sequencing to analyze global differential gene expression in a defined model nerve cell line expressing α4β2 nicotinic receptors (nAChRs), high-affinity targets for beta amyloid (Aβ). The nAChR-expressing neuronal cells were treated with nanomolar Aβ 1-42 to gain insights into the molecular mechanisms underlying Aβ-induced neurotoxicity in the presence of this sensitizing target receptor. We identified 15 genes (out of 15,336) that were differentially expressed upon receptor-linked Aβ treatment. Genes up-regulated with Aβ treatment were associated with calcium signaling and axonal vesicle transport (including the α4 nAChR subunit, the calcineurin regulator RCAN3, and KIF1C of the kinesin family). Downregulated genes were associated with metabolic, apoptotic or DNA repair pathways (including APBA3, PARP1 and RAB11). Validation of the differential expression was performed via qRT-PCR and immunoblot analysis in the defined model nerve cell line and primary mouse neurons. Further verification was performed using immunocytochemistry. In conclusion, we identified apparent changes in gene expression on Aβ treatment in the presence of the sensitizing nAChRs, linked to early-stage Aβ-induced neurotoxicity, which may represent novel therapeutic targets. Amyloid-β (Aβ) is a short, potentially neurotoxic peptide derived from amyloid precursor protein (APP) in select regions of the brain 1,2. At "physiological" levels (pM), there is considerable evidence for Aβ functioning as a positive neuromodulator 3-7 , acting through neuronal signaling receptors. In Alzheimer's disease (AD), a progressive neurodegenerative disorder that is the most prevalent cause of dementia, histopathology is mainly characterized by extracellular plaques composed primarily of the Aβ peptide in fibrillar form, intracellular neurofibrillary tangles formed from hyperphosphorylated tau, and neuronal degeneration including extensive loss of cholinergic basal forebrain neurons. In addition, synaptic impairment and loss are central to changes in memory and cognition in AD 8. Notably, during the prodromic phase of AD, soluble oligomeric Aβ levels are dramatically increased (high nM to μM) years before diagnosis (see 9). There is ample evidence that it is the diffusible oligomeric Aβ assemblies that play a role in neurotoxicity 10 and contribute to driving development of synaptic impairment and degeneration, largely through induction of abnormal tau and, later, neuroinflammation 11,12. There remain important questions, however, in regard to the impact of elevated Aβ levels on neuronal function, integrity and viability, in particular altered signaling through known target receptors. Despite extensive understanding of the pathology of AD, differential diagnosis of the disease in the prodromic and early stages has been problematic, part...

Background Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by accumulation of extracellular amyloid beta (Aβ) and intracellular neurofibrillary tangles, leading to chronic activation of astrocytes and microglia and persistent neuroinflammation. Aβ-linked activation of microglia and astrocytes leads to increased intracellular calcium and production of proinflammatory cytokines, impacting the progression of neurodegeneration. An N-terminal Aβ fragment (Aβ1–15) and a shorter hexapeptide core sequence within the N-Aβ fragment (N-Aβcore: Aβ10–15) have previously been shown to protect against Aβ-induced mitochondrial dysfunction, oxidative stress and apoptosis in neurons and rescue synaptic and spatial memory deficits in an APP/PSEN1 mouse model. Here, we hypothesized that the N-Aβ fragment and N-Aβcore are protective against Aβ-induced gliotoxicity, promoting a neuroprotective environment and potentially alleviating the characteristically persistent neuroinflammation present in AD. Methods We treated ex vivo organotypic brain slice cultures from an aged familial AD mouse model, 5xFAD, with the N-Aβcore and used immunocytochemistry to assess the impact on astrogliosis and microgliosis and alterations in synaptophysin-positive puncta engulfed by microglia. Isolated neuron/glia cultures, mixed glial cultures or a microglial cell line were treated with oligomeric human Aβ at concentrations mimicking the pathogenic concentrations (μM) observed in AD in the absence or presence of the non-toxic N-terminal Aβ fragments. Resultant changes in synaptic density, gliosis, oxidative stress, mitochondrial dysfunction, apoptosis, and the expression and release of proinflammatory markers were then determined. Results We demonstrate that the N-terminal Aβ fragments mitigated the phenotypic switch leading to astrogliosis and microgliosis induced by pathological concentrations of Aβ in mixed glial cultures and organotypic brain slice cultures from the transgenic 5xFAD mouse model, while protecting against Aβ-induced oxidative stress, mitochondrial dysfunction and apoptosis in isolated astrocytes and microglia. Moreover, the addition of the N-Aβcore attenuated the expression and release of proinflammatory mediators in microglial cells activated by Aβ and rescued microglia-mediated loss of synaptic elements induced by pathological levels of Aβ. Conclusions Together, these findings indicate the protective functions of the N-terminal Aβ fragments extend to reactive gliosis and gliotoxicity induced by Aβ, by preventing or reversing glial reactive states indicative of neuroinflammation and synaptic loss central to AD pathogenesis.