Institution: NIH Library Sign In as Member / Individual

This Article Full Text Services Alert me to new issues of the journal Request Copyright Permission

Total Chemical Synthesis. RANTES (CCL5) analogs were prepared by polymer-supported organic synthesis of two fragments, XYZ-RANTES(2-33)-thioester (where XYZ is a code representing the nonamino acid N-terminal substituent of the analog) and RANTES (Cys³⁴-Ser⁶⁸). t-Butoxycarbonyl (Boc) chemistry was used, as described in ref. 1. The fragments, when deprotected and cleaved from the resin, were purified and coupled in a native chemical ligation (2). Where XYZ was an acyl group, the 1-hydroxy-7-azabenzotriazole (HOAt) esters of the relevant acids were applied to the N-terminal fragment while still on the resin. n-Nonyl-des-Ser¹()-RANTES (NNA-RANTES) was prepared by reaction of RANTES(2-33)-thioester-resin with 2 M 1-bromo-n-nonane in dimethylformamide (DMF) and three equivalents of cesium carbonate at 40°C for 8 h. Hexyl-[Gly¹]RANTES (HEA-[Gly¹]RANTES) was prepared by reacting the RANTES(2-33)-thioester-resin with the symmetrical anhydride of bromoacetic acid, followed by the addition of n-hexylamine. n-Dodecanoyl-RANTES(3-68) (DDY-RANTES) was prepared by coupling n-dodecanoic acid as the HOAt ester to RANTES(3-33)-thioester-resin. All other methods were as described in refs. 1 and 3.

Preparation of CD4 T Cells for CC-Chemokine Receptor 5( CCR5) Down-Modulation Assay. Human peripheral blood mononuclear cells (PBMC) were obtained from healthy adult volunteers after informed consent. PBMC were activated with phytohemagglutinin-P (PHA-P) and IL-2 as described (4). Activated cells were cultured for 10-14 days to maximize CCR5 expression (4). CD4 T cells were purified by antibody-mediated removal of other PBMC subsets.

HIV Replication Assay. Activated PBMC were infected with the CCR5-tropic HIV-1 strain BaL in the presence of serial dilutions of chemokines. HIV-1 replication after several days of culture was measured by p24 capsid protein ELISA (4).

Activity of Lead Molecules in Virus-Based Assays. The RANTES analogs fell broadly into the same rank order of potency identified in the cell fusion assay (PSC-RANTES>NNY-RANTES>AOP-RANTES>Met-RANTES» CAP-RANTES; see Fig. 5), with similar proportional increases in potency between AOP-RANTES, NNY-RANTES, and PSC-RANTES. The absolute potencies were generally lower than those found in the cell fusion assay, with IC₅₀ values for the more potent analogs » 10-fold higher. Among possible explanations for this are: (i) use of a viral strain expressing a different R5-tropic envelope to that used in the cell fusion assay (BaL rather than ADA), (ii) use of target cells that may exhibit different sensitivity to RANTES analogs (PBMC rather than HeLa-P4-CCR5 cells), and (iii) any effects of the analogs on aspects of the viral life cycle other than the attachment/fusion step.

1. Wilken, J., Hoover, D., Thompson, D. A., Barlow, P. N., McSparron, H., Picard, L., Wlodawer, A., Lubkowski, J. & Kent, S. B. (1999) Chem. Biol. 6, 43-51.

2. Dawson, P. E., Muir, T. W., Clark-Lewis, I. & Kent, S. B. (1994) Science 266, 776-779.

3. Wilken, J. & Kent, S. B. (1998) Curr. Opin. Biotechnol. 9, 412-426.

4. Sabbe, R., Picchio, G. R., Pastore, C., Chaloin, O., Hartley, O., Offord, R. & Mosier, D. E. (2001) J. Virol. 75, 661-671.