Phage randomization in a charybdotoxin scaffold leads to CD4-mimetic recognition motifs that bind HIV-1 envelope through non-aromatic sequences

Binding of HIV-1 gp120 to T-cell receptor CD4 initiates conformational chan ges in the viral envelope that trigger viral entry into host cells. Phage e pitope randomization of a p-turn loop of a charybdotoxin-based miniprotein scaffold was used to identify peptides that can bind gp120 and block the gp 120-CD4 interaction. We describe here the display of the charybdotoxin scaf fold on the filamentous phage FUSE5, its use to construct a p-turn library, and miniprotein sequences identified through library panning with immobili zed Env gp120. Competition enzyme-linked immunosorbent assay (ELISA) identi fied high-frequency phage selectants for which specific gp120 binding was c ompeted by sCD4. Several of these selectants contain hydrophobic residues i n place of the Phe that occurs in the gp120-binding B-turns of both CD4 and previously identified scorpion toxin CD4 mimetics. One of these selectants , denoted TXM[(24)GQTL(27)], contains GQTL in place of the CD4 beta -turn s equence (40)QGSF(43) TXM[(24)GQTL(24)] peptide was prepared using solid-pha se chemical synthesis, its binding to gp120 demonstrated by optical biosens or kinetics analysis and its affinity for the CD4 binding site of gp120 con firmed by competition ELISA. The results demonstrate that aromatic-less loo p-containing CD4 recognition mimetics can be formed with detectable envelop e protein binding within a p-turn of the charybdotoxin miniprotein scaffold . The results of this work establish a methodology for phage display of a c harybdotoxin miniprotein scaffold and point to the potential value of phage -based epitope randomization of this miniprotein for identifying novel CD4 mimetics. The latter are potentially useful in deconvoluting structural det erminants of CD4-HIV envelope recognition and possibly in designing antagon ists of viral entry.