Functional Characterization Of Human CYPJ, And Screening For Low Molecular Inhibitors Of CYPJ And CYPA

Cyclophilin belongs to the peptidyl-prolyl cis-trans isomerase(PPIase)superfamily. The PPIase activity of cyclophilin can be inhibited by the widely usedimmunoimpressive agent cyclosporin A (CsA). Cyclophilins are evolutionarilyconserved protein, which widely expressed in most organisms such as plant,bacteria, insets, fungus, and mammals. In recent years, many important roles ofcyclophilin have been discovered in the pathological processes of apoptosis, tumor,immflamation, and virus infection.By electronic differencial display techniques, we have identified a novelcDNA up-regulated in hepatocellular carcinoma (HCC). This novel member ofcyclophilin protein family was named cyclophilin J (CYPJ) by HUGO. Weexamined the expression of CYPJ in 56 pairs of cancer/non-cancerous tissuesamples, and found its mRNA was elevated in 71.42% samples tested. Therecombinant CYPJ contains PPIase activity, and can be inhibited by cyclosporin A.Over-expression of CYPJ promote growth and clonal formation of L02 andSK-Hepl cells in vitro, and promote the growth of liver cancer cells in athymicnude mice model in vivo. Based on these findings, we further evaluated the roles ofCYPJ in HCC. We generated lentivirus containing sh-RNA targeting CYPJ toknockdown its expression. Compared with control virus, the sh-RNA containinglentivirus can repress cancer cell growth in vitro and in vivo. The mechanism forCYPJ to promote cell growth is probably related to its characteristics to regulatecell cycle. Transient CYPJ transfection reduce G₁ phase cell populations andincrease S phase populations; on the contrary, knockdown the expression of CYPJby siRNA or inhibit its activity by CsA result in increased G₁ phase cells andreduced S phase cell populations. This phenomenon indicates that CYPJ canpromote the transition of cell populations from G₁ phase to S phase. CYPJover-expression elevates the expression of cyclin D1, and Knockdown theexpression of CYPJ by siRNA or inhibit its PPIase activity by CsA reduces theexpression of cyclin D1. In the cyclin D1 promoter luciferase reporter assay, it is found that the transcriptional regulation by CYPJ depends on its PPIase activity,and the pathway may mediaed by multiple transcriptional factors.At the mean time, we performed structure based screening of low molecularweight ligands of CYPA/CYPJ. We carried out computer-aided drug screening, andfound several ligands targeting CYPA/CYPJ. Then we conformed the binding byBIACORE, and their inhibitory activities were evaluated by PPIase assay. Finally,we got 12 compounds with inhibitory activities against CYPA/CYPJ, and theanti-HIV-1 activities of these compounds were also tested. Five compounds (FD5,FD8, FD9, FD10, and FD12) can inhibit over 50% of HIV-1 replication at theconcentration of 10μM. At the mean time, we examined the inhibitory activity ofFD12 on the growth of several HCC cell lines. The further works of structure andactivity relationship analysis (SAR) is going on.In summary, we found that knockdown the expression of CYPJ by RNAi orinhibit its activity by CsA can withdraw the growth of liver cancer cells. We alsofound that CYPJ can promote the transition of cell cyle from G1 phase to S phase,and this process is related to regulation of cyclin D1. This work provides a usefulcandidate gene for carcinogenesis, and provide new clue for novel diagnostic andtherapeutic agent. We also screened several inhibitors of CYPA and CYPJ based ontheir structures. Among all these compounds, five can dramatically inhibit thereplication of HIV-1, and one can inhibit tumor growth. These inhibitors providenew method to study the roles of cyclophilin in virus replication and tumorgenesis.At the mean time, they can be modificated to develop more potent anti-HIV andanti-tumor drugs.