Interaction of human cyclophilin hCyp-18 with short peptides suggests the existence of two functionally independent subsites.

The binding of peptides, derived from the model substrate Suc-Ala-Ala-Pro-Phe-pNA, to the human cyclophilin hCyp-18 was investigated. HCyp-18 is able to bind 2-4-mer peptides as well as shorter para-nitroaniline (pNA) derivatives and pNA surrogates. Although Suc-Ala-Phe-pNA binds hCyp-18, only proline-containing peptides are able to block efficiently the peptidyl-prolyl cis/trans isomerase activity. Competition experiments strongly suggest the existence of two independent subsites: a S1' 'proline' subsite and a S2'-S3' 'pNA' subsite. The interaction at S2'-S3' requires either a Phe-pNA C-terminus or a Phe-pNA surrogate bearing an H-bond acceptor able to bind Trp121 and Arg148 simultaneously.

Design of a Gag pentapeptide analogue that binds human cyclophilin A more efficiently than the entire capsid protein: new insights for the development of novel anti-HIV-1 drugs.

Cyclophilin A (hCyp-18), a ubiquitous cytoplasmic peptidyl-prolyl cis/trans isomerase (PPIase), orchestrates HIV-1 core packaging. hCyp-18, incorporated into the virion, enables core uncoating and RNA release and consequently plays a critical role in the viral replication process. hCyp-18 specifically interacts with a single exposed loop of the Gag polyprotein capsid domain via a network of nine hydrogen bonds which mainly implicates a 7-mer fragment of the loop. As previously reported, the corresponding linear heptapeptide Ac-Val-His-Ala-Gly-Pro-Ile-Ala-NH(2) (2) binds to hCyp-18 with a low affinity (IC(50) = 850 +/- 220 microM) but a potentially useful selectivity for hCyp-18 relative to hFKBP-12, another abundant PPIase. On the basis of X-ray structures of Gag fragments:hCyp-18 complexes, we generated a series of modified peptides in order to probe the determinants of the interaction and hence to select a peptidic ligand displaying a higher affinity than the capsid domain of Gag. We synthesized a series of heptapeptides to test the energetic contribution of amino acids besides the Gly-Pro moiety. In particular the importance of the histidine residue for the interaction was underscored. We also investigated the influence of N- and C-terminal modifications. Hexapeptides containing either deaminovaline (Dav) in place of the N-terminal valine or substitution of the C-terminal alanine amide with a benzylamide group displayed increased affinities. Combination of both modifications gave the most potent competitor Dav-His-Ala-Gly-Pro-Ile-NHBn (28) which has a higher affinity for hCyp-18 (K(d) = 3 +/- 0.5 microM) than the entire capsid protein (K(d) = 16 +/- 4 microM) and a very low affinity for hFKBP-12. Some of our results strongly suggest that the title compound is not a substrate of hCyp-18 and interacts preferentially in the trans conformation.