Improving the Anti-HIV Potency of Different Compounds through Synergy and Covalent Linkage: Dimerization Studies of CXCL8

Abstract

In the first part of my dissertation we focused on the development of covalently linking compounds that bind gp120 with those that bind gp41 in order to block HIV fusion. We used griffithsin or CD4M33, that both bind to gp120, covalently linked with C-peptide C37 of gp41. The results show the linked compound Griff37 is several-fold more potent than griffithsin alone in both fusion and viral assay, making Griff37 an attractive candidate for further development as a microbicide against HIV. In the second part of my dissertation we investigate the effect of combining HIV fusion inhibitors having differing mechanisms of action. We used P2-RANTES and RANTES that both bind to the chemokine receptor CCR5 on the surface of the human cells along with C-peptides C37 and C34 that bind the viral envelop glycoprotein gp41. We found that the combination of RANTES with C37/C34 has an additive effect on fusion assay. In contrast P2-RANTES and C37/C34 shows synergy in inhibition of cell fusion. In viral assays using MAGI cells the synergy between P2-RANTES and C37/C34 is minimal. In the third part of my dissertation we focused on characterization of two examples of evolutionarily conserved unfavorable sequence motifs that affect quaternary structure. In contrast to the straightforward action of favorable sequences, these unfavorable motifs produce interactions disfavoring one outcome to indirectly promote another one. To identify such motifs, we propose and developed a statistically validated computational method combining structure and phylogeny. This approach was applied in an analysis of the alternate forms of homodimerization exhibited in the chemokine family. We identified two evolutionarily conserved sequence motifs in the CC subfamilies: a drastic two-residue deletion (ERV) and a simple point mutation (V27R). Cloned into the CXCL8 background, these two motifs were experimentally proven to confer a monomeric state. NMR analyses indicate that these variants are structured in solution and retain the chemokine fold. Structurally, the motifs retain a chemokine tertiary fold while introducing unfavorable quaternary interactions that inhibit CXCL8 dimerization.