Inhibition of Human Immunodeficiency Virus Type 1 Entry by a Keggin Polyoxometalate

Wang, Xiaoli; Wang, Jiao; Zhang, Wenmei; Li, Boye; Zhu, Ying; Hu, Qin; Yang, Yishu; Zhang, Xiaoguang; Yan, Hong; Zeng, Yi

Surface plasmon resonance (SPR) binding assay was performed using BIAcore 3000 (GE Healthcare) [32] . The surface of the CM5 sensor chip was activated by injecting 0.05 M N-hydroxysuccimide (NHS) and 0.2 M N-ethyl-N -(diethylaminopropyl)-carbodiimide (EDC) at a flow rate of 10 µL/min for 10 min. The N36 peptide (5 µM), CD4 recombinant protein (0.5 µM), and gp120 (0.5 µM) recombinant protein were dissolved in 10 mM acetate buffer (pH 5), respectively and immobilized to chips using amine coupling. The binding sites were then blocked with a 1.0 M ethanolamine-HCl (pH 8.5) injection. In the direct binding assay, PT-1 of diluted serial concentrations was injected over the surface of N36 peptide and CD4 protein at the rate of 10 µL/min for 10 min. In the competition assay, 0.7 µM PT-1 were incubated with 1.1 µM soluble CD4 protein for 15 min prior to injection. Then, 2 µM PT-1, 1.1 µM CD4 and the PT-1/CD4 mixture were injected to the flow cell over the gp120 protein, respectively. In both assays, the unmodified surface was used as a reference surface. The resonance unit value (RU) was measured for data analysis. The equilibrium constant for dissociation (KD) values was determined using the BIAvaluation software (GE Healthcare).