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The chemokine receptor CCR5 is utilized as a critical coreceptor by most primary HIV-1 strains. While the lack of structural information on CCR5 has hampered the rational design of specific inhibitors, mimetics of the chemokines that naturally bind CCR5 can be molecularly engineered. We used a structure-guided approach to design peptide mimetics of the N-loop and β1-strand regions of regulated on activation normal T-cell-expressed and secreted (RANTES)/CCL5, which contain the primary molecular determinants of HIV-1 blockade. Rational modifications were sequentially introduced into the N-loop/β1-strand sequence, leading to the generation of mimetics with potent activity against a broad spectrum of CCR5-specific HIV-1 isolates (IC(50) range: 104-640 nM) but lacking activity against CXCR4-specific HIV-1 isolates. Functional enhancement was initially achieved with the stabilization of the N loop in the β-extended conformation adopted in full-length RANTES, as confirmed by nuclear magnetic resonance (NMR) analysis. However, the most dramatic increase in antiviral potency resulted from the engraftment of an in silico-optimized linker segment designed using de novo structure-prediction algorithms to stabilize the C-terminal α-helix and experimentally validated by NMR. Our mimetics exerted CCR5-antagonistic effects, demonstrating that the antiviral and proinflammatory functions of RANTES can be uncoupled. RANTES peptide mimetics provide new leads for the development of safe and effective HIV-1 entry inhibitors.

Background: Purification of polypeptides and proteins derived from recombinant DNA techniques and of long synthetic polypeptides often represents a challenge. Affinity methods exist, but generally require addition of a large recognition unit to the target protein and use of expensive purification media. Use of large units is dictated by the characteristics of non-covalent complexes, where the energy necessary to form the complex derives from the sum of multiple weak energy interactions. Covalent interactions in contrast are of high energy, even when only a few bonds are formed. We decided to explore the use of the reversible covalent bond formed between N-terminal cysteine and threonine residues with an aldehyde as a method of protein purification., Results: A series of test peptides with N-terminal cysteine and threonine were captured by a polyethyleneglycol-polyacrylamide resin to which an aldehyde function had been grafted. Peptides with other amino acids at the N-terminus did not interact with the resin. A recombinant polypeptide with N-terminal cysteine was purified to 90% purity in one step. Polypeptides were eluted from the resin simply by adding a hydroxylamine derivative, which reacts with aldehyde functions to form an oxime., Conclusions: Polypeptides possessing N-terminal cysteine or threonine can be easily purified using this 'covalent capture' approach.