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Sphingopeptides: dihydrosphingosine-based fusion inhibitors against wild-type and enfuvirtide-resistant HIV-1.

ABSTRACT: Understanding the structural organization of lipids in the cell and viral membranes is essential for elucidating mechanisms of viral fusion that lead to entry of enveloped viruses into their host cells. The HIV lipidome shows a remarkable enrichment in dihydrosphingomyelin, an unusual sphingolipid formed by a dihydrosphingosine backbone. Here we investigated the ability of dihydrosphingosine to incorporate into the site of membrane fusion mediated by the HIV envelope (Env) protein. Dihydrosphingosine as well as cholesterol, fatty acid, and tocopherol was conjugated to highly conserved, short HIV-1 Env-derived peptides with no antiviral activity otherwise. We showed that dihydrosphingosine exclusively endowed nanomolar antiviral activity to the peptides (IC(50) as low as 120 nM) in HIV-1 infection on TZM-bl cells and on Jurkat T cells, as well as in the cell-cell fusion assay. These sphingopeptides were active against enfuvirtide-resistant and wild-type CXCR4 and CCR5 tropic HIV strains. The anti-HIV activity was determined by both the peptides and their dihydrosphingosine conjugate. Moreover, their mode of action involved accumulation in the cells and viruses and binding to membranes enriched in sphingomyelin and cholesterol. The data suggest that sphingopeptides are recruited to the HIV membrane fusion site and provide a general concept in developing inhibitors of sphingolipid-mediated biological systems.

SUBMITTER: Ashkenazi A

PROVIDER: S-EPMC3475257 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The peptide drug enfuvirtide (T20) is the only viral fusion inhibitor used in combination therapy for HIV-1 infection, but it has relatively low antiviral activity and easily induces drug resistance. Emerging studies demonstrate that lipopeptide-based fusion inhibitors, such as LP-11 and LP-19, which mainly target the gp41 pocket site, have greatly improved antiviral potency and in vivo stability. In this study, we focused on developing a T20-based lipopeptide inhibitor that lacks pocket-binding sequence and targets a different site. First, the C-terminal tryptophan-rich motif (TRM) of T20 was verified to be essential for its target binding and inhibition; then, a novel lipopeptide, termed LP-40, was created by replacing the TRM with a fatty acid group. LP-40 showed markedly enhanced binding affinity for the target site and dramatically increased inhibitory activity on HIV-1 membrane fusion, entry, and infection. Unlike LP-11 and LP-19, which required a flexible linker between the peptide sequence and the lipid moiety, addition of a linker to LP-40 sharply reduced its potency, implying different binding modes with the extended N-terminal helices of gp41. Also, interestingly, LP-40 showed more potent activity than LP-11 in inhibiting HIV-1 Env-mediated cell-cell fusion while it was less active than LP-11 in inhibiting pseudovirus entry, and the two inhibitors displayed synergistic antiviral effects. The crystal structure of LP-40 in complex with a target peptide revealed their key binding residues and motifs. Combined, our studies have not only provided a potent HIV-1 fusion inhibitor, but also revealed new insights into the mechanisms of viral inhibition.IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection; however, T20 requires high doses and has a low genetic barrier for resistance, and its inhibitory mechanism and structural basis remain unclear. Here, we report the design of LP-40, a T20-based lipopeptide inhibitor that has greatly improved anti-HIV activity and is a more potent inhibitor of cell-cell fusion than of cell-free virus infection. The binding modes of two classes of membrane-anchoring lipopeptides (LP-40 and LP-11) verify the current fusion model in which an extended prehairpin structure bridges the viral and cellular membranes, and their complementary effects suggest a vital strategy for combination therapy of HIV-1 infection. Moreover, our understanding of the mechanism of action of T20 and its derivatives benefits from the crystal structure of LP-40.

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Sphingopeptides: dihydrosphingosine-based fusion inhibitors against wild-type and enfuvirtide-resistant HIV-1.

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