Anti-HIV cyclotides from the Chinese medicinal herb Viola yedoensis

Abstract

Cyclotides are macrocyclic plant peptides characterized by a knotted arrangement of three disulfide bonds. They display a range of interesting bioactivities, including anti-HIV and insecticidal activities. More than 100 different cyclotides have been isolated from two phylogenetically distant plant families, the Rubiaceae and Violaceae. In this study we have characterized the cyclotides from Viola yedoensis, an important Chinese herb from the Violaceae family that has been reported to contain potential anti-HIV agents. From V. yedoensis five new and three known cyclotides were identified and shown to have anti-HIV activity. The most active of these is cycloviolacin Y5, which is one of the most potent of all cyclotides tested so far using in vitro XTT-based anti-HIV assays. Cycloviolacin Y5 is the most hydrophobic of the cyclotides from V. yedoensis. We show that there is a positive correlation between the hydrophobicity and the anti-HIV activity of the new cyclotides and that this trend tracks with their ability to disrupt membranes, as judged from hemolytic assays on human erythrocytes.

Figure 1.

Sequence and structure (PDB ID: 1NB1) of the prototypic cyclotide kalata B1. Cyclotides have a well-defined three-dimensional structure, which is stabilized by a cystine knot and a cyclic peptide backbone. Cyclotides have six loops and six Cys residues, labeled I-VI. Panel A shows the sequence and disulfide connectivity of kalata B1. The numbering begins at the Gly residue in loop 6 and proceeds in a clockwise direction. The three-dimensional structure of kalata B1 is illustrated in panel B, with β-strands indicated with darkened arrows (also highlighted on the sequence in panel A).

Figure 2.

LC-MS profile and sequence of cyclotides characterized from V. yedoensis. Panel A shows the LC-MS profile of the crude extract run at a 1%/min gradient of solvent 2 (90% CH₃CN, 0.1% HCO₂H): solvent 1 (0.1% HCO₂H) starting at 15% solvent 2. Each major peak is labeled sequentially according to elution time, and the characterized cyclotides are labeled in bold. The sequences of the identified cyclotides are shown in panel B. Peak 3 was difficult to purify and gave multicomponent MS/MS spectra, which were difficult to interpret, and it was not examined further. Cyclotides have three disulfide bonds, and the disulfide connectivity is likely to be conserved across all cyclotides. The disulfide connectivity of the prototypic cyclotide, kalata B1, is shown in Figure 1.

Figure 3.

Hemolytic activity of cyclotides from V. yedoensis. Hemolytic activities of kalata B1, cycloviolacin Y1, cycloviolacin Y4, cycloviolacin Y5, and melittin were measured for human erythrocytes. The HD₅₀ values (and 95% confidence intervals) were calculated using Prism software to be 11.7 μM (10.2–13.4 μM) for kalata B1, 9.3 μM (7.7–11.1 μM) for cycloviolacin Y4, 8.7 μM (7.4–10.2 μM) for cycloviolacin Y5, and 0.94 μM (0.90–0.97 μM) for melittin. Melittin is a well-known hemolytic agent that was used as a positive control and gave an activity consistent with literature values.

Figure 4.

Effect of hydrophobicity on cyclotide bioactivity. The anti-HIV activities (panels A and B) and hemolytic activities (panels B and C) of kalata B1, varv E, cycloviolacin Y1, cycloviolacin Y4, and cycloviolacin Y5 were correlated with hydrophobicity, which was measured experimentally using RP-HPLC (retention times from Figure 2) and theoretically using surface area calculations. The surface hydrophobicity was calculated by modeling the cyclotides using MODELLER.

Figure 5.

Surface representations of selected cyclotides from V. yedoensis. Surface representations are shown for cycloviolacin Y1 (A), kalata B1 (B), and cycloviolacin Y5 (C). Hydrophobic residues (Ala, Leu, Ile, Val, Trp, Phe, Pro) are shaded black.