2001;507:39. operating with mechanisms of action different from those of the above inhibitors are sought. HIV-1 integrase (IN) is a critical enzyme for the stable infection of host cells since it catalyzes the insertion of viral DNA into the genome of host cells, CNT2 inhibitor-1 by means of strand transfer and 3-end processing reactions and thus it is an attractive target for the Rabbit polyclonal to NPSR1 development of anti-HIV agents. Recently, the first IN inhibitor, raltegravir (Merck),2 has appeared in a clinical setting. It is assumed that the activity of IN must be negatively regulated during the translocation of the viral DNA from the cytoplasm to the nucleus to prevent auto-integration. The virus, as well as the host cells, must encode mechanism(s) to prevent auto-integration since the regulation of IN activity is critical for the virus to infect cells.3 By screening a library of overlapping peptides derived from HIV-1 SF2 gene products we have found three Vpr-derived peptides, 1, 2 and 3, which possess significant IN inhibitory activity, indicating that IN inhibitors exist in the viral pre-integration complex (PIC).4 The above inhibitory peptides, 1, 2 and 3, are consecutive overlapping peptides (Figure 1). Compounds 4 and 5 are 12- and 18-mers from the original Vpr sequence with the addition of an octa-arginyl group5 into the C-terminus for cell membrane permeability, respectively. Compounds 4 and 5 have IN inhibitory activity and anti-HIV activity. Here we report structure-activity relationship studies on these lead compounds for the development of more potent IN inhibitors. Open in a separate window Figure 1 Amino acid sequences of compounds 1C5. Compounds 1C3 are consecutive overlapping peptides with free N-/C-terminus. These were found by the IN inhibitory screening of a peptide library derived from HIV-1 gene products. Compounds 4 and 5 are cell penetrative leads of IN inhibitors. 2. Results and discussion To determine which lead compound is most suitable for further experiments, five peptides 6C10, which were elongated by one amino acid starting with compound 4 and extended ultimately to 5, were synthesized (Figure 2). Judging by the 3-end processing and strand transfer reactions strain C41. The solubility of the mutant protein was examined in a crude cell lysate, as follows. Cells were grown in 1 L of culture medium containing 100 g/mL of ampicillin at 37C until the optical density of the culture at 600 nm was between 0.4 and 0.9. Protein expression was induced by the addition of isopropyl-1-thio–D-galactopyranoside to a final concentration of 0.1 mM. After 2 h, the cells were collected by centrifugation at 6,000 rpm for 30 min. After removal of the supernatant, the cells were resuspended in HED buffer (20 mM HEPES, pH 7.5, 1 mM EDTA, 1 mM DTT) with 0.5 mg/mL lysozyme and stored on ice for 30 min. The cells were sonicated until the solution exhibited minimal viscosity then it was centrifuged at 15,000 rpm for 30 min. After removal of the supernatant, the pellet was dissolved in TNM buffer (20 mM Tris/HCl, pH 8.0, 1 M NaCl, 2 mM 2-mercaptoethanol) with 5 mM imidazole and stored on ice for 30 min. The cells were then centrifuged at 15,000 rpm for 30 min and the supernatant was collected. The supernatant was then filtered through 0.45 m filter cartridge and applied to a HisTrap column at 1 mL/min flow rate. After loading, the column was washed with 10 volume of TNM buffer with 5 mM imidazole. Protein was eluted with a linear gradient of 500 mM imidazole, containing TNM buffer. Fractions containing IN were pooled and checked with SDS-PAGE. 4.3 CD spectroscopy of peptides with Glu-Lys substitution CD measurements were performed on a JASCO J720 spectropolarimeter equipped with thermo-regulator (JASCO Corp., Ltd.), using 5 M of peptides dissolved in 0.1 M phosphate buffer, pH 5.6 containing 50% MeOH. UV spectra were recorded at 25 C in a quartz cell 1.0 mm path length, a time constant of 1 1 s, and a 100.340. different from those of the above inhibitors are sought. HIV-1 integrase (IN) is a critical enzyme for the stable infection of host cells since it catalyzes the insertion of viral DNA into the genome of host cells, by means of strand transfer and 3-end processing reactions and thus it is an attractive target for the development of anti-HIV agents. Recently, the first IN inhibitor, raltegravir (Merck),2 has appeared in a clinical setting. It is assumed that the activity of IN must be negatively regulated during the translocation of the viral DNA from the cytoplasm to the nucleus to prevent auto-integration. The virus, as well as the host cells, must encode mechanism(s) to prevent auto-integration since the regulation of IN activity is critical for the virus to infect cells.3 By screening a library of overlapping peptides derived from HIV-1 SF2 gene products we have found three Vpr-derived peptides, 1, 2 and 3, which possess significant IN inhibitory activity, indicating that IN inhibitors exist in the viral pre-integration complex (PIC).4 The above inhibitory peptides, 1, 2 and 3, are consecutive overlapping peptides (Figure 1). Compounds 4 and 5 are 12- and 18-mers from the original Vpr sequence with the addition of an octa-arginyl group5 into the C-terminus for cell membrane permeability, respectively. Compounds 4 and 5 have IN inhibitory activity and anti-HIV activity. Here we report structure-activity relationship studies on these lead compounds for the development of more potent IN inhibitors. Open in a separate window Figure 1 Amino acid sequences of compounds 1C5. Compounds 1C3 are consecutive overlapping peptides with free N-/C-terminus. These were found by the IN inhibitory screening of a peptide library derived from HIV-1 gene products. Compounds 4 and 5 are cell penetrative leads of IN inhibitors. 2. Results and discussion To determine which lead compound is most suitable for further experiments, five peptides 6C10, which were elongated by one amino acid starting with compound 4 and extended ultimately to 5, were synthesized (Figure 2). Judging by the 3-end processing and strand transfer reactions strain C41. CNT2 inhibitor-1 The solubility of the mutant protein was examined in a crude cell lysate, as follows. Cells were grown in 1 L of culture medium containing 100 g/mL of ampicillin at 37C until the optical density of the culture at 600 nm was between 0.4 and 0.9. Protein expression was induced by the addition of isopropyl-1-thio–D-galactopyranoside to a final concentration of 0.1 mM. After 2 h, the cells were collected by centrifugation at 6,000 rpm for 30 min. After removal of the supernatant, the cells were resuspended in HED CNT2 inhibitor-1 buffer (20 mM HEPES, pH CNT2 inhibitor-1 7.5, 1 mM EDTA, 1 mM DTT) with 0.5 mg/mL lysozyme and stored on ice for 30 min. The cells were sonicated until the solution exhibited minimal viscosity then it was centrifuged at 15,000 rpm for 30 min. After removal of the supernatant, the pellet was dissolved in TNM buffer (20 mM Tris/HCl, pH 8.0, 1 M NaCl, 2 mM 2-mercaptoethanol) with 5 mM imidazole and stored on ice for 30 min. The cells were then centrifuged at 15,000 rpm for 30 min and the supernatant was collected. The supernatant was then filtered through 0.45 m filter cartridge and applied to a HisTrap column at 1 mL/min flow rate. After loading, the column was washed with 10 volume CNT2 inhibitor-1 of TNM buffer with 5 mM imidazole. Protein was eluted with a linear gradient of 500 mM imidazole, containing TNM buffer. Fractions containing IN were pooled and checked with SDS-PAGE. 4.3 CD spectroscopy of peptides with Glu-Lys.