Acetylshikonin attenuates angiotensin II-induced proliferation and motility of human brain smooth muscle cells by inhibiting Wnt/β-catenin signaling

Cerebrovascular smooth muscle cell proliferation and migration contribute to hyperplasia in case of cerebrovascular remodeling and stroke. In the present study, we investigated the effects of acetylshikonin, the main ingredient of a Chinese traditional medicine Zicao, on human brain vascular smooth muscle cell (HBVSMCs) proliferation and migration induced by angiotensin II (AngII), and the underlying mechanisms. We found that acetylshikonin treatment significantly inhibited AngII-induced HBVSMCs proliferation and cell cycle transition from G1 to S phase. Wound-healing assay and Transwell assay showed that AngII-induced cell migration and invasion were markedly attenuated by acetylshikonin. In addition, AngII challenge significantly induced Wnt/β-catenin signaling activation, as evidenced by increased β-catenin phosphorylation and nuclear translocation and GSK-3β phosphorylation. However, acetylshikonin treatment inhibited the activation of Wnt/β-catenin signaling. Consequently, western blotting analysis revealed that acetylshikonin effectively reduced the expression of downstream target genes in AngII-treated cells, including c-myc, survivin and cyclin D1, which contributed to the inhibitory effect of acetylshikonin on HBVSMCs proliferation. Further, stimulation with recombinant Wnt3a dramatically reversed acetylshikonin-mediated inhibition of proliferation and cell cycle transition in HBVSMCs. Our study demonstrates that acetylshikonin prevents AngII-induced cerebrovascular smooth muscle cells proliferation and migration through inhibition of Wnt/β-catenin pathway, indicating that acetylshikonin may present a potential option for the treatment of cerebrovascular remodeling.     

Rab21, a Novel PS1 Interactor,Regulates γ-Secretase Activity via PS1 Subcellular Distribution

γ-Secretase has been a therapeutical target for its key role in cleaving APP to generate β-amyloid (Aβ), the primary constituents of senile plaques and a hallmark of Alzheimer’s disease (AD) pathology. Recently, γ-secretase-associating proteins showed promising role in specifically modulating APP processing while sparing Notch signaling; however, the underlying mechanism is still unclear. A co-immunoprecipitation (Co-IP) coupled with mass spectrometry proteomic assay for Presenilin1 (PS1, the catalytic subunit of γ-secretase) was firstly conducted to find more γ-secretase-associating proteins. Gene ontology analysis of these results identified Rab21 as a potential PS1 interacting protein, and the interaction between them was validated by reciprocal Co-IP and immunofluorescence assay. Then, molecular and biochemical methods were used to investigate the effect of Rab21 on APP processing. Results showed that overexpression of Rab21 enhanced Aβ generation, while silencing of Rab21 reduced the accumulation of Aβ, which resulted due to change in γ-secretase activity rather than α- or β-secretase. Finally, we demonstrated that Rab21 had no effect on γ-secretase complex synthesis or metabolism but enhanced PS1 endocytosis and translocation to late endosome/lysosome. In conclusion, we identified a novel γ-secretase-associating protein Rab21 and illustrate that Rab21 promotes γ-secretase internalization and translocation to late endosome/lysosome. Moreover, silencing of Rab21 decreases the γ-secretase activity in APP processing thus production of Aβ. All these results open new gateways towards the understanding of γ-secretase-associating proteins in APP processing and make inhibition of Rab21 a promising strategy for AD therapy.     

Novel naftopidil derivatives containing methyl phenylacetate and their blocking effects on α1D/1A-adrenoreceptor subtypes

α₁-Adrenoceptor (α₁-AR) antagonists are considered to be the most effective monotherapy agents for lower urinary tract symptoms associated with benign prostatic hyperplasia (LUTS/BPH). In this study, we synthesized compounds 2–17, which are novel piperazine derivatives that contain methyl phenylacetate. We then evaluated the vasodilatory activities of these compounds. Among them, we found that compounds 2, 7, 12, which contain 2-OCH₃, 2-CH₃ or 2, 5-CH₃, respectively, exhibited potent α₁-blocking activity similar to protype drug naftopidil (1). The antagonistic effects of 2, 7, and 12 on the (?)-noradrenaline-induced contractile response of isolated rat prostatic vas deferens (α_1A), spleen (α_1B) and thoracic aorta (α_1D) were further characterized to assess the sub receptor selectivity. Compared with naftopidil (1) and terazosin, compound 12 showed the most desirable α_1D/1A subtype selectivity, especially improved α_1A subtype selectivity, and the ratios pA₂ (α_1D)/pA₂ (α_1B) and pA₂ (α_1A)/pA₂ (α_1B) were 17.0- and 19.5-fold, respectively, indicating less cardiovascular side effects when used to treat LUTS/BPH. Finally, we investigated the chiral pharmacology of 12. We found, however, that the activity of enantiomers (R)-12 and (S)-12 are not significantly different from that of rac-12.     

Discovery and optimization of novel constrained pyrrolopyridone BET family inhibitors

Novel conformationally constrained BET bromodomain inhibitors have been developed. These inhibitors were optimized in two similar, yet distinct chemical series, the 6-methyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-ones (A) and the 1-methyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-ones (B). Each series demonstrated excellent activity in binding and cellular assays, and lead compounds from each series demonstrated significant efficacy in in vivo tumor xenograft models.     

Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors

Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity.     

Functional analysis of Cullin 3 E3 ligases in tumorigenesis

Cullin 3-RING ligases (CRL3) play pivotal roles in the regulation of various physiological and pathological processes, including neoplastic events. The substrate adaptors of CRL3 typically contain a BTB domain that mediates the interaction between Cullin 3 and target substrates to promote their ubiquitination and subsequent degradation. The biological implications of CRL3 adaptor proteins have been well described where they have been found to play a role as either an oncogene, tumor suppressor, or can mediate either of these effects in a context-dependent manner. Among the extensively studied CRL3-based E3 ligases, the role of the adaptor protein SPOP (speckle type BTB/POZ protein) in tumorigenesis appears to be tissue or cellular context dependent. Specifically, SPOP acts as a tumor suppressor via destabilizing downstream oncoproteins in many malignancies, especially in prostate cancer. However, SPOP has largely an oncogenic role in kidney cancer. Keap1, another well-characterized CRL3 adaptor protein, likely serves as a tumor suppressor within diverse malignancies, mainly due to its specific turnover of its downstream oncogenic substrate, NRF2 (nuclear factor erythroid 2-related factor 2). In accordance with the physiological role the various CRL3 adaptors exhibit, several pharmacological agents have been developed to disrupt its E3 ligase activity, therefore blocking its potential oncogenic activity to mitigate tumorigenesis.