1. The Screen And Functional Studies Of Cell Cycle Inhibitors With Budding Yeast As A Model Organism

Cell cycle is a collection of many tightly regulated events that contribute to cellduplication. Deregulation of these events will result in abnormal cell division thatleads to unfaithful DNA duplication, inaccurate chromosome segregation, or celldeath. The accumulation of mutations may result in uncontrolled cell growth, ahallmark of cancer cells. On the other hand, inhibition of some critical cell cycleevents will block cell growth, a common strategy for cancer treatment.Cell cycle progression is driving by cyclin-dependent kinases (CDKs). CDKsphosphorylate their targets and promote DNA synthesis and chromosome segregation.Thus CDK is a potential antitumor target. CDKs are regulated by either associationwith positive regulatory subunits known as cyclins, or negative regulators known asCDK inhibitors (CKIs). The balance of these regulations controls CDK activity andcoordinate various cell cycle processes. The protein levels of Cyclins and CKIs aretightly regulated both transcriptionally and post-translationally. The combination oftranscription and ubiquitin-mediated proteolysis ensures that some cell cycleregualtors appear at a specific cell cycle stage.The ubiquitin-proteasome pathway (UPP) is the main protein degradationmechanism that mediates the substrate-specific timely degradation of unstable cellcycle regulators. The SCF complex (composed of Skp1, Cdc53 (Cullin) and F-boxproteins) is an E3 enzyme required for degradation of G1 cyclins and CDK inhibitorSic1. Mutations in SCF complex block cell cycle at G1 phase, indicating the role ofSCF in G1-S transition. Budding yeast is an excellent modal system to study the cellcycle regulation. Because most of the cell cycle regulators are conserved from yeast tohuman, the research with budding yeast will benefit the understanding of cell cycleregulation in human cells. In this study we used the temperature sensitivity mutants cdc53-1 and skp1 toscreen SCF inhibitors from a compound library and the collection of fermentationbroths. Three positive fermentation broths and one compound (DH334) were obtainedfrom this screen. The growth inhibition of DH334 to wild type, cdc53-1 mutant andsic1 mutant was determined with disc paper, FACS and budding index. We found thatDH334 could inhibit the growth of budding yeast. Strikingly, deletion of SIC1, whichencodes the CDK inhibitor, resulted in resistance to DH334. In contrast, cdc53-1mutant cells defective for Sic1 degradation exhibited more pronounced sensitivity toDH334. The presence of DH334 caused accumulation of yeast cells in G₁ phase. Theeffect of DH334 on the degradation of Sic1 protein and the phosphorylation of Sld2 (ayeast CDK substrate) were examined using Western-Blotting. We found that thedegradation of Sic1 protein was not inhibited by DH334, while Sld2 phosphorylationdelayed significantly in the presence of DH334. FACS was used to detect the cellcycle inhibition of HepG2, SGC, and HEL cancer cell lines and we found that DH334also blocked mammalian cell cycle in G₁ phase. The inhibition of mammalian CyclinA-CDK by DH334 was determined in vitro and we found that the IC₅₀ of the DH334was 27μM. The growth inhibition of mammalian cell lines by 12 beta-carbolinederivatives was studied with MTT and the IC₅₀, the growth inhibition of mammaliancells by the tested derivatives showed some correlation with their toxicity to yeastcells.In summary our studies have provided strong evidence indicating that buddingyeast could a valuable model system for the screen anticancer drugs. Also, we can usebudding yeast as model system to study the molecular mechanisms of anticancerdrugs. This work lays the foundation for our further screen for novel cell cycleinhibitors that could be used as anticancer drugs. The discovery of antimicrobial drugs has made it possible to control thebacterial infections. However, the control of virus infection is still a challenge due tothe lack of efficient and specific antivirus drugs. Although hundreds of antivirus drugshave been discovered and 40 of them are used clinically, these drugs either haverelative low bioavailability, high toxicity to human cells, or easily become resistant.Thus the search for new antivirus drugs is still an important task in the 21 century forscientists.In this work, an antiviral substance, named JN219, was isolated from fungalmetabolites. The extracorporeal antivirus method was used to detect the antivirusactivity of JN219 on many viruses including HCMV, HSV-1, HSV-2, RSV, Ad3, COXB1-6, VSV, RV AS11, and 15 influenza viruses. JN219 showed strong antivirusactivity towards HCMMV, VSV, and RV. It also exhibited weak antivirus activitytowards HSV-1, HSV-2, and RSV. However, no obvious antivirus activity towardsAD3 and COX-B1-6 was noticed. We used HCMV as a model system to investigatethe antivirus mechanism and found that JN219 could detain the irruption andamalgamation of HCMV. By using a mouse model, we found that JN219 couldalleviate diarrhea symptom induced by RV (SA11) infection. Compared with theinfected mice, JN219 prevents the fall off mucous membrane. Moreover, no abnormalexudation was observed in the enteric cavity in JN219 treated animals. The stabilitystudies indicate that the EC₅₀of JN219 drops from 0.4μg/ml to 39.7μg/ml after 30days and the activity decreased 99.25 times.