YU Zhiling (Ph.D.) 禹志领 博士

1. 2006 World Congress on Chinese Medicine Developing the next generation of anticancer agents from Chinese medicinal herbs using DNA replication-initiation proteins as the targets YU Zhiling (Ph.D.) 禹志领 博士

2. Outline • Discovery of DNA replication-initiation proteins as novel anticancer drug targets. • Development of an anticancer drug screening platform with the interaction of DNA replication-initiation proteins as the target. • Identification of anticancer agents from Chinese medicinal herbs.

3. Novel Targets

4. Initiator ORC (1-6): MCM (2-7): Origin Recognition Complex Minichromosome Maintenance CDC (6, 45): Others: NOC3, CDT1,MCM10, SLD2,3 Cell Division Cycle The Initiation of DNA Replication Other Initiation Proteins Replicator Origin Cdt1 Cdc6 ARS1 MCM ORC Cdc45 Noc3 B3 B2 B1 A

5. DNA Replication-initiation Proteins are Associated with Cancer • Every cancer cell must replicate its DNA, which requires the function of initiation proteins, in order to divide. • DNA replication initiation proteins are highly expressed in cancer cells but not in normal non-proliferating cells. Therefore, these proteins are attractive markers for cancer detection. • In the human body, more than 90% cells are non-proliferating. • Inhibiting the expression of DNA replication-initiation proteins can induce cancer cells to undergo apoptosis but without affecting the viability of normal cells. Therefore, DNA replication initiation proteins are attractive targets of anticancer drugs.

6. Phase TUNEL Liver Cancer Cells Normal Liver Cells The Antisense Oligonucleotide Induces Apoptosis in Cancer but not Normal Cells Inhibiting the expression of DNA replication-initiation proteins induces apoptosis in human cancer cells.

7. The Antisense Oligonucleotides Reduce Tumor Size in Nude Mice 4000 3500 3000 2500 2000 1500 Oligo: 2 µg/g/day 1000 500 Day 0 DNA replication-initiation proteins are attractive anticancer drug targets. US patent, Sep. 2002.

8. Novel Drug Screening Platform

9. Anticancer Drug Screening Platforms Using MCM proteins as the targets • The coupling of expression of the MCM proteins with proliferation offers the potential of identifying drugs with low toxicity. • Their essential roles in proliferation make them effective targets for drugs to kill cancer cells with high potency and broad spectrum. • Disruption of the MCM complex by drug candidates can be easily detected in the reverse yeast two-hybrid system.

10. Patterns of proliferating cells in normal and malignant tissue from the cervix or colon. malignant normal cervix colon Immunostained with antibodies against MCM5 (brown) .

11. Mcm2-7p MCM Proteins • The MCM protein family, with six members (Mcm2p-7p), was originally identified in yeast from mutants defective in minichromosome maintenance (MCM) and is required for the initiation of DNA replication. • They exist in vivo as a hexamer containing every subunit and function in the form of the heterohexamericcomplex.

12. Co-IP from Crosslinked Human Cell Protein Extracts -M7 Bead MCMx MCM7 Mcm7p interacts with Mcm3p but not Mcm2p

13. Mcm7 Mcm4 Mcm6 Mcm3 Mcm2 Mcm5 Pairwise Interactions of Human MCM proteins • Yeast two-hybrid • GST-pull down • Co-IP from cross-linked human cells

14. An Anticancer Drug Screening Platform: The Reverse Yeast Two-Hybrid System

15. Characteristics of the Novel Platform • Identifying low toxicity anticancer drugs • Inhibiting the target proteins will kill cancerous but not normal proliferating calls • The target proteins are highly expressed in proliferating but not in quiescent cells • The novel platform screens for agents that can make cells grow • Identifying high potency anticancer drugs • The target proteins are essential for DNA replication and cell proliferation • Identifying broad-spectrum anticancer drugs • A cancer cell of any kind and at any stag needs to replicate its DNA for dividing

16. New Generation Botanical Drugs?

17. Anticancer TCM Screening Grow yeast cells in 96 well plates; add TCM Cell growth No growth Potential anti-cancer drugs The compounds cannot disrupt the interaction of the two proteins Test in human cells and animals Seven hundred extracts, fractions or compounds isolated from Chinese medicines have been screened A few samples were found to be anticancer drug candidates.

18. A Candidate compound made the reverse two-hybrid yeast cells grow in the screening assays (values are the mean ± SD, n = 5, DMSO is the solvent)

19. AA389 (10 μg/mL in DMSO) killed HeLa cells UT DMSO AA389 * 0.774 ± 0.089 0.768 ± 0.099 0.187 ± 0.018 * MTT assays: OD570, mean ± SD, n = 6

20. 286 UT UT DMSO DMSO L-02 (Normal liver) HepG2 (Liver cancer) 286

21. Summary • Discovered novel cancer drug targets. • Established a novel cancer drug screening platform. • Identified a few candidate botanical anticancer agents. • New generation anticancer drugs?

22. Acknowledgments Professors in BICH & BIOL departments of HKUST Lab mates in Prof. ChunLiang’s Lab at HKUST Co-workers in Dr. Zhihong Jiang’s Lab @ HKBU Co-workers in Prof. Wen-luan Wendy Hsiao’s Lab @ HKBU Co-workers in Prof. Wang-fun David FONG’s Lab @ HKBU Group members: An Cheng, Guan Yongwen, Sharon Kwok Sum Ling