Novel cathepsin K inhibitors block osteoclasts in vitro and increase spinal bone density in zebrafish.

Cathepsin K (Cat K) is a predominant cysteine protease and highly potent collagenase expressed in osteoclasts. Cat K inhibitors are anti-resorptive agents to treat osteoporosis. A novel scaffold of cathepsin K inhibitors, exemplified by lead compound 1x, was used as the template for designing and synthesizing a total of 61 derivatives that have not been reported before. An exploratory structure-activity relationship analysis identified the potent Cat K inhibitor A22, which displayed an IC50 value of 0.44 µM against Cat K. A22 was very specific for Cat K and caused a significantly higher in vitro inhibition of the enzyme as compared to that of lead compound 1x. A surface plasmon resonance analysis confirmed in vitro binding of A22 to Cat K. Molecular docking studies indicated several favourable interaction sites for A22 within the active pocket of Cat K. Furthermore, A22 also blocked active osteoclasts in vitro and increased spinal bone density in zebrafish, in which it showed an activity that was higher than that of the marketed therapeutic bone metabolizer etidronate disodium. A22 represents a very promising lead compound for the development of novel antiresorptive agents functioning as orthosteric inhibitors of Cat K.

[Huotan Jiedu Tongluo decoction alleviates early atherosclerosis of rabbits by inhibiting eNOS uncoupling pathway].

The aim of this paper was to study the curative effect of Huotan Jiedu Tongluo (HTJDTL) decoction on a rabbit model with early atherosclerosis (AS),and furtherly to explore whether it could inhibit the BH4/eNOS uncoupling ROS or not. Twenty-four Japanese white rabbits were randomly divided into sham operation group, model group, HTJDTL decoction group and atorvastatin group. Rabbit models with early atherosclerosis were established by high fat diet, nitrogen drying and carotid artery balloon injury. The rabbits were sacrificed at 7th days after balloon injury and several parameters were measured. The pathological morphology of the common carotid artery was observed by HE staining. The blood lipids were detected by peroxidase method. The ratio of vascular eNOS dimer and monomer was measured by Western blot. The ELISA and biochemical technology were respectively used for testing BH4 and ROS levels in serum. The results showed that compared with the sham operation group, the model group had mild stenosis of the common carotid artery lumen, uneven intimal hyperplasia, lipid deposition in the intima and media, and obvious hyperplasia of the adventitia with inflammatory cell infiltration. The HTJDTL decoction could significantly inhibit the intimal hyperplasia compared with the model group, meanwhile, reduce the lipid deposition of the media and the infiltration of the adventitial cells. Compared with the sham operation group, the blood lipids and ROS of the model animals significantly increased, but BH4 and the ratio of eNOS dimer/monomer decreased. Compared with the model group, HTJDTL decoction significantly reduced the TC, ox-LDL and ROS levels, and also up-regulated eNOS dimer/monomer ratio, but it increased BH4 trend without statistical difference. According to the results, it was found that HTJDTL decoction couldsignificantly prevent and improve the vascular remodeling of rabbits model with early atherosclerosis. The mechanism of decoction may largely be related to the inhibition of BH4/eNOS uncoupling and the reduction of oxidative stress.

[Evaluation of osteogenic effects of BMP2 up-regulator E40071 in vitro].

Bone morphogenetic protein 2(BMP2) plays a key role in bone development and reestablishment. In the study, we screened up-regulators of BMP2 among 20 000 compounds through a cell-based high throughput screening model and a positive compound E40071 [2-(4-(5-methyl-3-phenylpyrazolo[1,5-a]pyrimidin-7-yl) piperazin-1-yl)ethan-1-ol] was found as the positive hit. The EC(50) value of E40071 was 2.73 µmol·L(-1). In vitro, E40071 upregulated the m RNA levels of BMP2 and the downstream transcription factors, Runx2 and Osx in MC3T3-E1(subclone 14). Protein expression of Runx2 was up-regulated by E40071 through induction of Smad1/5/8 phosphorylation. The alkaline phosphatase(ALP) activity was increased by E40071. Moreover, E40071 promoted the mineralization of MC3T3-E1(subclone 14) by Alizarin red S staining. In addition, E40071 markedly inhibited osteoclast differentiation of mice macrophage Raw264.7 induced by RANKL and reduced the expression of osteoclast differentiation markers, including MMP9 and NFATc1. The results suggest that E40071 is able to promote bone formation activity of osteoblasts and inhibit differentiation of osteoclasts.

[In vitro effect and mechanistic study of compound E23869 on lipid metabolism].

This study was designed to identify inducers of ATP-binding cassette transporter A1(ABCA1) and CD36 and lysosomal integral membrane protein-II analogous-1(CLA-1) and to evaluate the in vitro effect of the active compound on lipid metabolism. Among 20 000 compounds screened, E23869 was found as a positive hit using cell-based high throughput screening models. The up-regulating activities of E23869 in ABCA1p-LUC and CLA-1p-LUC Hep G2 cells were 196% and 198%, respectively. The EC(50) values of E23869 in ABCA1p- LUC and CLA-1p-LUC Hep G2 cells were 0.25 µmol·L(-1) and 0.66 µmol·L(-1), respectively. E23869 significantly upregulated the protein levels of ABCA1, scavenger receptor class B type I(SR-BI)/CLA-1 and ATP-binding cassette transporter G1(ABCG1) in both macrophages RAW264.7 and L02 cells by Western blotting analysis. Foam cell assay showed that E23869 inhibited lipids accumulations in macrophages RAW264.7. Cholesterol efflux assay showed that E23869 induced HDL-mediated cholesterol efflux in macrophages RAW264.7. Moreover, E23869 up-regulated ABCA1, SR-BI/CLA-1 and ABCG1 expressions through activation of PPARα and PPARγ. In addition, E23869 weakly promoted in vitro differentiation of mouse preadipocytes 3T3-L1. In conclusion, E23869 up-regulated ABCA1, SR-BI/CLA-1 and ABCG1 expressions to promote cholesterol efflux, which is a good leading compound for regulation of lipid metabolism.