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

Abstract

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.

Altered HIV-1 Gag Protein Interactions with Cyclophilin A (CypA) on the Acquisition of H219Q and H219P Substitutions in the CypA Binding Loop

“…We also found that these substitutions reduced CypA incorporation into virions (Fig. 3C), which is compatible with previous reports by two groups (28,29). It was therefore postulated that MT-2 and H9 cells contained high CypA amounts, thereby compromising HIV-1 WT replication.…”

Dynamic Combinatorial Self‐Assembly of Cyclophilin hCyp‐18 Ligands through Oxorhenium Coordination

“…Complexation yields varied only slightly upon addition of recombinant hCyp-18 (final concentration: 64 mm), [9] except in the case of complex 4 i, which was formed more efficiently when cyclophilin was added to the mixture (Figure 2 A, graphs a and b), an effect that we called the "cyclophilin-enhancing effect". As expected, all complexes readily dissociated upon addition of GSH in the absence of hCyp-18 (Figures 2 B and D, graph d).…”

Refinement and 3D‐QSAR Studies of Inhibitors of Cyclophilin A Containing Amide Linker

“…However, inhibitors of CypA are mainly derived from the nature sources (such as CsA [2], FK506 [10], rapamycin [11], and sanglifehrin A [12]) and peptide analogs [13], which are all structural complex molecules, and little has been reported regarding the small molecule CypA inhibitors. Recently, using a strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and bioassay, we have designed and synthesized several series of novel small molecular CypA inhibitors, 17 of which showed PPIase inhibitory activities with IC 50 values at micromolar level [14 -17].…”