Design of a novel cyclotide-based CXCR4 antagonist with anti-human immunodeficiency virus (HIV)-1 activity

Abstract

Herein, we report for the first time the design and synthesis of a novel cyclotide able to efficiently inhibit HIV-1 viral replication by selectively targeting cytokine receptor CXCR4. This was accomplished by grafting a series of topologically modified CVX15 based peptides onto the loop 6 of cyclotide MCoTI-I. The most active compound produced in this study was a potent CXCR4 antagonist (EC50≈20 nM) and an efficient HIV-1 cell-entry blocker (EC50≈2 nM). This cyclotide also showed high stability in human serum, thereby providing a promising lead compound for the design of a novel type of peptide-based anticancer and anti-HIV-1 therapeutics.

Figure 1

Design of MCoTI-based cyclotides to target the cytokine receptor CXCR4. A. Primary and tertiary structures of cyclotide MCoTI-I. Structure is based on a homology model using the solution structure of MCoTI-II as template (PDB: 1IB9). The backbone cyclized peptide (connecting bond shown in green) is stabilized by the three-disulfide bonds (shown in red). The residues used for the grafting of a CVX15-based peptide are shown in blue on the structure and sequence of MCoTI-I. B. Sequence and co-crystal structure of peptide CVX15 bound to cytokine receptor CXCR4 (PDB: 3OE0). Peptide CVX15 is shown as a ribbon representation in green with the side-chains of the Cys residues involved in the disulfide bond in ball-and-stick form. The solvent accessible surface of the binding site of CXCR4 is shown in grey. C. Scheme depicting the approach used to design the different MCo-CVX cyclotides. A circularly permuted version of CVX15 was grafted onto loop 6 of MCoTI-I at different residues. The CVX15-based insert was created by joining the C and N-terminus directly through a flexible Gly_n linker and opening the new sequence at the D-Pro-Pro segment. Residues in red denote mutations or extra Gly residues introduced to increase flexibility. Single letter codes B, X and p represent the amino acid, 2-naphthylalanine, citruline and D-proline, respectively. Molecular graphics were built with Yasara (www.yasara.org).

Figure 2

Chemical synthesis and characterization of cyclotide MCo-CVX-5c. A. Analytical HPLC traces of the linear thioester precursor, GSH-induced cyclization/folding crude after 96 h and purified cyclotide. An arrow indicates the desired peptide. B. ES-MS characterization of pure MCo-CVX-5c. The expected average molecular weight is shown in parenthesis. C. Chemical shifts differences of the backbone, NH and H^α protons between the common sequence (residues 1 through 29) of MCoTI-I^, and MCo-CVX-5c (Table S2).

Figure 3

Biological characterization of MCo-CVX cyclotides. A. Competitive inhibition of SDF1α-mediated CXCR4 activation by different cyclotides. The peptide CVX15 Gln6Cit and the small molecule CXCR4 antagonist AMD3100 were used as controls. The assay was performed using CXCR4-bla U2OS cells. B. Inhibition of Erk phosphorylation (residues Thr²⁰² and Tyr²⁰⁴) by cyclotide MCo-CVX-5c. Cyclotide MCoTI-I and peptide CVX15 Gln6Cit were used as negative and positive controls, respectively. Erk phosphorylation was visualized by Western blot using CaOV3 cells treated with increasing amounts of CXCR4 inhibitor in the presence of SDF1α. C. Dose response inhibition of HIV-1 replication in MT-4 cells by cyclotides MCoTI-I and MCo-CVX-5c. The peptide CVX15 Gln6Cit and the small molecule HIV-1 integrase inhibitor, Raltegravir, were used as positive controls. Cyclotide MCoTI-I was used as negative control. The average of standard deviation of three experiments is shown. NB and ND stand for not bound and not determined, respectively.