In vitro activity: AMD-070 is active against X4 strain of HIV-1, HIV-1 IIIb in MT-4 cells, and The IC50 values for AMD-070 are 9-fold higher (0.009 μM vs 0.001 μM) and 8.7-fold higher (0.003 μM vs 0.026 μM) in PBMCs compared to MT-4 cells. AMD-070 has antiviral ability with the IC50 value of 15.5 nM.

Kinase Assay: SUP-T1 T cells are first preincubated with the compounds (with 1 as a control) for 30 min on ice, washed with PBS with 2% FCS, and incubated with PE-conjugated anti-CXCR4 mAb for 30 min on ice. After being washed with PBS, the cell samples are fixed with 1% paraformaldehyde in PBS and analyzed on a FACS Calibur flow cytometery. The dose-dependent inhibitory effects of the compounds on mAb binding are determined using the mean fluorescence intensity values.

Cell Assay: he activated cells (PHA-stimulated blasts) are washed three times with PBS, and viral infections are done. HIV-infected or mock-infected PHA-stimulated blasts are cultured in the presence of 25 U/mL of IL-2 and varying concentrations of compounds. Supernatant is collected at day 10, and HIV-1 core antigen in the culture supernatant is analyzed by the p24 viral Ag ELISA kit. Inhibition of HIV-1 replication in MT-4 cells is performed as previously described. Anti-HIV-1 activity and cytotoxicity measurements are carried out in parallel. They are based on the viability of MT-4 cells that has been infected with HIV-1 in the presence of various concentrations of the test compounds. The ICsub>50 is defined as the concentration required to inhibit 50% of the virus-infected cells against viral cytopathicity.