Hao Wang

We have discovered a potent DYRK1A inhibitor Y16-5 by virtual screening and in vitro biological evaluation. This compound showed strong in vitro neuroprotective activity and favorable druglikeness.

Background: Alzheimer’s disease (AD) is a neurodegenerative disease characterized by memory deficit and has emerged as a growing global health concern. Phosphodiesterase-8 (PDE8) is a cyclic adenosine monophosphate (cAMP)-specific hydrolase and its correlation with AD pathogenesis remains underexplored. Here, the effects and mechanisms of PF-04957325 (denoted as PF), a PDE8 inhibitor, were investigated in reversing AD both in vitro and in vivo. Methods: Briefly, BV2 cells were incubated with amyloid-β oligomers (AβO) to construct an AD cell model. Then, 2-month-old male C57BL/6J mice injected with AβO into the hippocampus and 10-month-old male amyloid precursor protein/presenilin-1 (APP/PS1) mice were used to construct AD animal models. Cells and mice were treated with PF to observe the effects of PDE8 on behavior and pathology related to AD. The Y-maze, novel object recognition (NOR), and Morris water maze (MWM) were performed to investigate cognitive function in mice. Western blot and immunofluorescence staining were used to identify the microglial activation state. Lastly, Western blot and ELISA were conducted to determine the levels of inflammatory factors and the proteins of PDE8/cAMP/CREB signaling. Results: PF-04957325 pretreatment reversed the conversation of proinflammatory microglia in BV2 cells induced by AβO, while also suppressing the levels of inflammatory factors, including interleukin-1β, interleukin-6, tumor necrosis factor-α, inducible nitric oxide synthase , and cyclooxygenase-2. In addition, AβO incubation upregulated the expression of PDE8 and concurrently downregulated that of brain-derived neurotrophic factor (BDNF), cAMP, p-PKA/PKA, and p-CREB/CREB in BV2 cells, all of which were reversed by PF. In vivo experiments evidenced impaired performance in the Y-maze, NOR, and MWM; these effects were reversed by PF. Similarly, PF treatment significantly attenuated microglia activation and the release of the inflammatory factors, and reversed the changes in the expression of BDNF and PDE8/cAMP/CREB signaling in AD mice. Finally, PF reduced the generation of Aβ1-42 by suppressing the expression of APP and PS1 in APP/PS1 mice. Conclusions: PF alleviated AD-like changes in behavior and pathology through various mechanisms, including attenuating microglia-mediated neuroinflammation, upregulating the expression of BDNF, restoring synaptic dysfunction, and inhibiting Aβ generation, which appear to be involved by PDE8/cAMP/CREB signaling. These results highlight the therapeutic potential of targeting PDE8 inhibition for AD treatment.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is implicated in accumulation of amyloid β-protein (Aβ) and phosphorylation of Tau proteins, and thus represents an important therapeutic target for neurodegenerative diseases. Though many DYRK1A inhibitors have been discovered, there is still no marketed drug targeting DYRK1A. This is partly due to the lack of effective and safe chemotypes. Therefore, it is still necessary to identify new classes of DYRK1A inhibitors. By performing virtual screening with the workflow mainly composed of pharmacophore modeling and molecular docking as well as the following DYRK1A inhibition assay, we identified compound L9, ((Z)-1-(((5-phenyl-1H-pyrazol-4-yl)methylene)-amino)-1H-tetrazol-5-amine), as a moderately active DYRK1A inhibitor (IC₅₀: 1.67 μM). This compound was structurally different from the known DYRK1A inhibitors, showed a unique binding mode to DYRK1A. Furthermore, compound L9 showed neuroprotective activity against okadaic acid (OA)-induced injury in the human neuroblastoma cell line SH-SY5Y by regulating the expression of Aβ and phosphorylation of Tau protein. This compound was neither toxic to the SH-SY5Y cells nor to the human normal liver cell line HL-7702 (IC₅₀: >100 μM). In conclusion, we have identified a novel DYRK1A inhibitor with neuroprotective activity through virtual screening and in vitro biological evaluation, which holds the promise for further study. Graphical abstract

Phosphodiesterase 8 (PDE8), as a member of PDE superfamily, specifically promotes the hydrolysis and degradation of intracellular cyclic adenosine monophosphate (cAMP), which may be associated with pathogenesis of Alzheimer’s disease (AD). However, little is currently known about potential role in the central nervous system (CNS). Here we investigated the distribution and expression of PDE8 in brain of mouse, which we believe can provide evidence for studying the role of PDE8 in CNS and the relationship between PDE8 and AD. Here, C57BL/6J mice were used to observe the distribution patterns of two subtypes of PDE8, PDE8A and PDE8B, in different sexes in vivo by western blot (WB). Meanwhile, C57BL/6J mice were also used to demonstrate the distribution pattern of PDE8 in selected brain regions and localization in neural cells by WB and multiplex immunofluorescence staining. Furthermore, the triple transgenic (3×Tg-AD) mice and wild type (WT) mice of different ages were used to investigate the changes of PDE8 expression in the hippocampus and cerebral cortex during the progression of AD. PDE8 was found to be widely expressed in multiple tissues and organs including heart, kidney, stomach, brain, and liver, spleen, intestines, and uterus, with differences in expression levels between the two subtypes of PDE8A and PDE8B, as well as two sexes. Meanwhile, PDE8 was widely distributed in the brain, especially in areas closely related to cognitive function such as cerebellum, striatum, amygdala, cerebral cortex, and hippocampus, without differences between sexes. Furthermore, PDE8A was found to be expressed in neuronal cells, microglia and astrocytes, while PDE8B is only expressed in neuronal cells and microglia. PDE8A expression in the hippocampus of both female and male 3×Tg-AD mice was gradually increased with ages and PDE8B expression was upregulated only in cerebral cortex of female 3×Tg-AD mice with ages. However, the expression of PDE8A and PDE8B was apparently increased in both cerebral cortex and hippocampus in both female and male 10-month-old 3×Tg-AD mice compared WT mice. These results suggest that PDE8 may be associated with the progression of AD and is a potential target for its prevention and treatment in the future.

Dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) plays an essential role in tau and Aβ pathology closely related to Alzheimer’s disease (AD). Accumulative evidence has demonstrated DYRK1A inhibition is able to reduce the pathological features of AD. Nevertheless, there is no approved DYRK1A inhibitors for clinical use as anti-AD drugs. This is somewhat the lack of effective and safe chemotypes of DYRK1A inhibitors. To address this issue, we carried out in silico screening, in vitro assays and in vivo efficacy evaluation with the aim to discover a new class of DYRK1A inhibitors for potential treatment of AD. By in silico screening, we selected and purchased 16 potential DYRK1A inhibitors from the Specs chemical library. Among them, compound Q17 (Specs ID: AO-476/40829177) potently inhibited DYRK1A. The hydrogen bonds between compound Q17 and each of three amino acid residues named GLU239, LEU241 and LYS188, were uncovered by molecular docking and molecular dynamics simulation. The cell-based assays showed that compound Q17 could protect SH-SY5Y cells from okadaic acid (OA)-induced injury by targeting DYRK1A. More importantly, compound Q17 significantly improved cognitive dysfunction in 3×Tg-AD mice, ameliorated pathological changes, and reduced the expression of DYRK1A, GSK-3β and GSK-3β (pSer9), attenuated tau hyperphosphorylation and Aβ deposition as well. In summary, our computational modeling strategy is effective to identify novel chemotypes of DYRK1A inhibitors with great potential to treat AD, and the identified compound Q17 in this study is worthy of further study. Graphic

Dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) is implicated in accumulation of amyloid β-protein (Aβ), phosphorylation of Tau proteins, and may eventually cause neurodegenerative diseases. Though many DYRK1A inhibitors have been discovered, there is still no marketed drug targeting DYRK1A. This is partly due to the lack of effective and safe chemotypes; therefore, it is still necessary to identify new classes of DYRK1A inhibitors. By the design of a virtual screening workflow composed of pharmacophore modeling and molecular docking and the following practical application, we identified compound L9, ((Z)-1-(((5-phenyl-1H-pyrazol-4-yl) methylene)-amino)-1H-tetrazol-5-amine), as a moderately active DYRK1A inhibitor (IC₅₀: 1.67 μM). This compound was structurally diverse compared with the known DYRK1A inhibitors, showed a unique binding mode to DYRK1A, and was not toxic to either SH-SY5Y cells or HL-7702 hepatocytes (IC₅₀＞100 μM). Furthermore, compound L9 showed neuroprotective activity by regulating the expression of Aβ and phosphorylation of Tau protein. In conclusion, we have identified a novel DYRK1A inhibitor through virtual screening and in vitro biological evaluation, which holds the promise for further study.

Depression is among the most frequent psychiatric comorbid conditions in Alzheimer disease (AD). However, pharmacotherapy for depressive disorders in AD is still a big challenge, and the data on the efffcacy of current antidepressants used clinically for depressive symptoms in patients with AD remain inconclusive. Here we investigated the mechanism of the interactions between depression and AD, which we believe would aid in the development of pharmacological therapeutics for the comorbidity of depression and AD. Female APP/PS1/Tau triple transgenic (3×Tg-AD) mice at 24 months of age and age- and sex-matched wild-type (WT) mice were used. The shuttle-box passive avoidance test (PAT) were implemented to assess the abilities of learning and memory, and the open field test (OFT) and the tail suspension test (TST) were used to assess depression-like behavior. High-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was used to detect the level of neurotransmitters related to depression in the hippocampus of mice. The data was identified by orthogonal projections to latent structures discriminant analysis (OPLS-DA). Most neurotransmitters exert their effects by binding to the corresponding receptor, so the expression of relative receptors in the hippocampus of mice was detected using Western blot. Compared to WT mice, 3×Tg-AD mice displayed significant cognitive impairment in the PAT and depression-like behavior in the OFT and TST. They also showed significant decreases in the levels of L-tyrosine, norepinephrine, vanillylmandelic acid, 5-hydroxytryptamine, and acetylcholine, in contrast to significant increases in 5-hydroxyindoleacetic acid, L-histidine, L-glutamine, and L-arginine in the hippocampus. Moreover, the expression of the alpha 1a adrenergic receptor (ADRA1A), serotonin 1 A receptor (5HT1A), and γ-aminobutyric acid A receptor subunit alpha-2 (GABRA2) was significantly downregulated in the hippocampus of 3×Tg-AD mice, while histamine H3 receptor (H3R) expression was significantly upregulated. In addition, the ratio of phosphorylated cAMP-response element-binding protein (pCREB) and CREB was significantly decreased in the hippocampus of 3×Tg-AD mice than WT mice. We demonstrated in the present study that aged female 3×Tg-AD mice showed depression-like behavior accompanied with cognitive dysfunction. The complex and diverse mechanism appears not only relevant to the imbalance of multiple neurotransmitter pathways, including the transmitters and receptors of the monoaminergic, GABAergic, histaminergic, and cholinergic systems, but also related to the changes in L-arginine and CREB signaling molecules.