Highly Potent Chimeric Inhibitors Targeting Two Steps of HIV Cell Entry

Bo Zhao², Marie K. Mankowski^†, Beth A. Snyder^†, Roger G. Ptak^†, and Patricia J. LiWang¹

¹University of California Merced, 5200 N. Lake Road, Merced, CA 95343

²Texas A&M University, Department of Biochemistry and Biophysics, College Station, TX 77843-2128

^†Southern Research Institute, Department of Infectious Disease Research, 431 Aviation Way, Frederick, Maryland 21701

Running Title: RANTES chimeras as potent HIV inhibitors.

Correspondence to: Patricia J. LiWang, Email: pliwang@ucmerced.edu, (209 228-4568)

Blocking HIV-1 cell entry has long been a major goal of anti-HIV drug development. Here we report a successful design of two highly potent chimeric HIV entry inhibitors composed of one CCR5-targeting RANTES variant (5P12-RANTES or 5P14-RANTES; (1)) linked to a gp41 fusion inhibitor, C37. Chimeric inhibitors 5P12-linker-C37 and 5P14-linker-C37 showed extremely high anti-viral potency in single-cycle and replication competent viral assays against R5 tropic viruses, with IC₅₀ values as low as 0.004 nM. This inhibition was somewhat strain dependent and was up to 100 fold better than the RANTES variant alone or in combination with unlinked C37. The chimeric inhibitors also fully retained the antiviral activity of C37 against X4 tropic viruses, and this inhibition can be further enhanced significantly if the target cell co-expresses CCR5 receptor. On human peripheral blood mononuclear cells, the inhibitors showed very strong inhibition against R5 tropic Ba-L strain and X4-tropic IIIB strain, with IC₅₀ values as low as 0.015 nM and 0.44 nM, which are 45 and 16 better than the parent inhibitors, respectively. A clear delivery mechanism requiring a covalent linkage between the two segments of the chimera was observed and characterized. Furthermore, the two chimeric inhibitors are fully recombinant and are easily produced at low cost. These attributes make them excellent candidates for anti-HIV microbicides. The results of this paper also suggest a potent approach for optimizing existing HIV entry inhibitors or designing new inhibitors.