Application of ring-closing metathesis macrocyclization to the development of Tsg101-binding antagonists

Viral release is a necessary component of HIV-1 replication. To be an efficient process, viral budding relies on recruitment of the ubiquitin E2 variant (UEV) domain of the human tumor susceptibility gene 101 protein (Tsg101) by major structural proteins of HIV-1.1,2 This entails the direct interaction of the Tsg101 UEV domain with a proline rich motif (PRM) within the viral Gagp6 protein that contains a conserved sequence, Pro-Thr/Ser-Ala-Pro [“P(T/S)AP”].3,4 Over-expression of the Tsg101 UEV inhibits virus release by interfering with processing of the p6 domains and this effect is abrogated by mutation within the P(T/S)AP binding site. In theory, blocking this critical Tsg101-p6 interaction could prevent viral budding and provide a basis for new targeted antiretroviral therapies.5,6 This is supported by the recent finding that inhibition of HIV budding can be achieved by cyclic peptides that interfere with Tsg101-Gag interactions.7 In an effort to develop Tsg101-binding inhibitors using the reported p6-derived 9-mer wild-type (WT) sequence “P1E2P3T4A5P6P7E8E9,” we had previously improved Tsg101-binding affinity by applying hydrazone- and hydrazide-library techniques.8 We also reported an oxime-based post solid-phase diversification approach to peptide library construction.9–11 However, the peptide mimetics resulting from this earlier work showed poor biovailability in cell-based experiments. Because poor cellular uptake was thought to contribute to the low bioavailability, peptides were conjugated with known membrane carrier antennapedia and HIV-Tat (48–60) peptides.12 Although these constructs did exhibit apparent enhanced cellular uptake, the Tsg101 binding affinity of the conjugates was seriously disrupted. Therefore, we sought an alternate approach that could improve cell bioavailability while maintaining Tsg101-binding affinity. We took note of the fact that cyclic versions of linear peptides can result in better cell-permeability. Additionally, appropriately restricting solution conformations through cyclization can also afford higher affinity.13–17 The objective of the current study was to apply RCM strategies to the WT 9-mer peptide and to observe the effects that such macrocyclization would have on Tsg101-binding affinity and cellular bioavailability.