Discovery Of Novel Small-molecule Human Vaccinia H1-related Phosphatase Inhibitors

Human vaccinia H1-related phosphatase (VHR) is a member of the atypical dual-specificity phosphatases. VHR is looked upon as most well characterized DSP in biochemical and kinetic studies since it consists of only a catalytic domain. VHR dephosphorylates and inactivates extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK), which are members of mitogen-activated protein kinases (MAPK). Recent studies show that VHR overexpresses in many tumor cell lines than corresponding primeray cell types, and plays a central role in regulating the progression of these tumor cells, such as cell growth, differentiation and senescence. VHR is a potential novel drug target for cancer therapy.Recombinant VHR were overexpressed in Escherichia coli and purified to homogeneous, and then the characteristics of active VHR were investigated, including the effects of DTT,EDTA and NaCl, pH dependence, the kinetics of enzyme reaction and inhibition by positive inhibitor(Na₃VO₄). By optimizing the reaction condition and screening system, we established the high-throughput screening (HTS) assay of VHR.After screening our chemical libraries containing 65,760 compounds, we got 34 potent inhibitors of VHR with IC₅₀ values lower than 3 ng/mL. Among them, Comp 1, with IC₅₀ value of 1.20μM, is a novel reversible and non-competitive inhibitor of VHR. The selectivity of Comp 1 on other PTPs was also tested. It did not show apparent inhibitory activity towards several receptor-type transmembrane protein tyrosine phosphatases such as CD45 (leukocyte common antigen) and LAR (leukocyte common antigen-related), and several non-receptor-type protein tyrosine phosphatases such as PTP1B (Protein Tyrosine Phosphatase 1B) and SHP1(SH2 domain containing tyrosine phosphase 1), and one dual-specificity phosphatase, CDC25A (cell division cycle 25 homolog A). And it showed weaker inhibition on SHP2 and CDC25B than VHR.We also evaluated the biological activity of Comp 1 on the cellular level. We found Comp 1 was able to enhance ERK and JNK phosphorylation in HeLa, and inhibit cell proliferation of HeLa, HT-29 and MDA-MB-435. Comp 1 can induce cell cycle arrest at G2/M phase and stimulate apoptosis in HeLa cells when in high concentrations.The discovery and characterization of the novel potent and selective inhibitor as well as its evaluation on the cellular level, will be ground work for further structure modification, cell-based and animal-based pharmacological and efficacy studies in the future. It might be a extremely powerful tool to exploit the physiological and pathological function of VHR.