Identification Of Inhibitor Of Human Dual-specific Phosphatase, VHR, By Computer-aided And NMR-based Screening

Our work focuses on identifying the inhibitor of Human Vaccina H1 related protein (VHR), by using virtual screening and NMR method. One compound with novel structure was found to be a competitive and potent inhibitor of VHR. And the effects of this inhibitor in cells were also investigated. VHR is a dual-specific phosphatase (DSPs) playing an important role in the mitogen-activated protein (MAP) kinase cascades regulation. It is also a potential drug target for designing inhibitors against diseases related to the immune response. The inhibitor may be contribute to the clarification of physiological function of VHR, and may be a promising candidate for designing of more effective agents against VHR.First chapter introduces and reviews the current docking methods in drug virtual screening. As a new method or technology for drug discovery, virtual screening has been involved into the pipeline of drug discovery and development as a practical tool. With the development of structural biology, structural genomics, functional genomics, and proteomics, more and more three dimensional structures of biologic molecule will be expressed, virtual screening would play a vital role which could accelerate the course of drug discovery.The second chapter is the brief review of NMR screening techniques in drug discovery and drug design. Library about NMR screening design is also discussed. Pharmaceutical and academic nuclear magnetic resonance (NMR) groups have implemented NMR screening techniques as a powerful approach to identify and to investigate protein/ligand interactions. Pharmaceutical groups in particular have incorporated NMR screening strategies into their drug discovery and drug design programs. This stems from the fact that NMR screening is naturally synergistic with combinatorial or medicinal chemistry, high throughput screening (HTS), structure-based drug design, and genomics.NMR screening can be defined as the identification of small molecule ligands for macromolecular targets by observation of a change in an NMR parameter that occurs upon their interaction. NMR screening methods can be divided into those that detect interactions by observation of either macromolecule NMR parameters or small molecule NMR parameters. In the case of macromolecules, the parameter that can be monitored is limited typically to chemical shifts. For small molecules, the choice of NMR parameters is more diverse. These include longitudinal, transverse; diffusion coefficients; and intermolecular and intramolecular magnetization transfer. The latter includes transferred NOE, NOE pumping and reverse NOE pumping, saturation transfer, and WaterLOGSY experiments.In the third chapter, we described the work about identifying the potent inhibitor of VHR. Human Vaccinia H1-related phosphatase (VHR) is a dual-specific phosphatase (DSPs) playing an important role in the mitogen-activated protein (MAP) kinase cascades regulation. It is also a potential drug target for designing inhibitors against diseases related to the immune response. Combining virtual and NMR-based ligand screening strategy, we successfully identified four VHR inhibitors, of which GATPT (glucosamine-aminoethoxy-triphenyl-tin) can bind to VHR with a K_i value of 2.54μM. The putative binding mode of GATPT was constructed by molecular docking simulation to provide structural insights into the ligand binding mechanism. Furthermore, we found that this compound can significantly inhibit the dephosphorylation of the extracellular regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs) and block transition of G1-S phase in the cell cycle. Therefore, GATPT structure is a promising candidate for designing of more effective agents against VHR.The fourth chapter introduced the work processed on identifying inhibitors of AF-6 PDZ domain. The human AF-6 gene was initially discovered as a fusion partner of the ALL-1 gene in acute lymphoblastic leukemia. The human AF-6, a scaffold protein between cell membrane-associated proteins and actin cytoskeleton, plays an important role in special cell-cell junctions and signal transduction. Molecules that can block cell adhesion may have value as therapeutic agents that prevent tumor metastasis. Therefore, the inhibitors of AF-6 PDZ domain may be a potential candidate for blocking cell adhesion and preventing tumor metastasis.