Grifonin-1: a small HIV-1 entry inhibitor derived from the algal lectin, Griffithsin

Abstract

Background: Griffithsin, a 121-residue protein isolated from a red algal Griffithsia sp., binds high mannose N-linked glycans of virus surface glycoproteins with extremely high affinity, a property that allows it to prevent the entry of primary isolates and laboratory strains of T- and M-tropic HIV-1. We used the sequence of a portion of griffithsin's sequence as a design template to create smaller peptides with antiviral and carbohydrate-binding properties.

Methodology/results: The new peptides derived from a trio of homologous β-sheet repeats that comprise the motifs responsible for its biological activity. Our most active antiviral peptide, grifonin-1 (GRFN-1), had an EC50 of 190.8±11.0 nM in in vitro TZM-bl assays and an EC(50) of 546.6±66.1 nM in p24gag antigen release assays. GRFN-1 showed considerable structural plasticity, assuming different conformations in solvents that differed in polarity and hydrophobicity. Higher concentrations of GRFN-1 formed oligomers, based on intermolecular β-sheet interactions. Like its parent protein, GRFN-1 bound viral glycoproteins gp41 and gp120 via the N-linked glycans on their surface.

Conclusion: Its substantial antiviral activity and low toxicity in vitro suggest that GRFN-1 and/or its derivatives may have therapeutic potential as topical and/or systemic agents directed against HIV-1.

Figure 1. Sequence of the griffithsin and comparison of the functional repeats (boxed) in its structure.

Position of the unknown amino acid X³¹ (shaded) is occupied by Ala in the expressed variant of the protein.

Figure 2. The structure of griffithsin.

Panel A: Dimer of griffithsin (PDB entry code 2GTY, [23]). Fragments corresponding to GRFN-1/2 and GRFN-3 are in red and in blue respectively. Remaining homological domain necessary to form core of the griffithsin's monomer is in green. Panel B: Core of griffithsin. Residues Tyr²⁸, Tyr⁶⁸ and Tyr¹¹⁰, which are components of monosaccharides' binding domain, are in magenta.

Figure 3. Analytical data for GRFN-1.

(A) analytical HPLC profile and (B) MS spectra.

Figure 4. Antiviral activity of GRFNs.

Panel A: Comparison of dose response experiments of GRFNs in TZM-bl assay. Panel B: Comparison of dose response experiments of GRFN-1 in TZM-bl assay (EC₅₀ = 190.8±11.0 nM) and p24^gag antigen release assay (EC₅₀ = 546.6±66.1 nM) with RC-101 in TZM-bl assay (EC₅₀ = 3404.0±91 nM). RC-101 is a θ-defensin which is currently being developed as topical microbicide. Panel C: Antiviral activity of griffithsin (GRFT) in TZM-bl assay (EC₅₀ = 19.6±1.9 pM). Panel D: Comparison of antiviral activity of GRFT and GRFN-1 in p24^gag antigen release assay using PBMCs and CXCR4 (HIV-1_IIIB) and CCR5 (HIV-1_BAL) strains.

Figure 5. Binding of GRFN-1 to viral glycoproteins.

Binding to: (A) gp120_LAV, (B) gp120_BAL, (C) gp41 and (D) GRFN-1 (self-association). K_D±SEM values were calculated as an average from at least 5 independent experiments.

Figure 6. Examples of SPR competition experiments of GRFN-1 with saccharide(s) using gp120_LAV chip.

(A)- galactose, (B) Man₃GlcNAc₂ (“core pentasaccharide”).

Figure 7. Stability of human RBCs in the presence of various concentrations of GRFN-1.

Figure 8. Viability (blue) and % of cell metabolism inhibition (red) of TZM-bl cells in the presence of various concentrations of GRFN-1.

Figure 9. Inhibition of cell metabolism by various concentrations of GRFN-1 (MTT assay) determined in primary vaginal epithelial cells (VEC), human peripheral blood mononuclear cells (PBMC) and PM1 cells (continuously CD4+ T-cell line, [52]).

Figure 10. Analysis of GRFN-1 structure.

(A) FTIR spectra; (B) CD spectra in in 10 mM phosphate buffer, pH = 6.5 ((---) 0.25 mM; (─) 0.5 mM); (C) CD spectra in TFE:10 mM phosphate buffer pH = 6.5 (4∶6, v∶v) and HFIP (---); 10 mM phosphate buffer pH = 6.5 (4∶6, v∶v) (─).

Figure 11. Evolution of GRFN-1 secondary structure as a function of simulation time in aqueous periodic solvent box.

Figure 12. Comparison of GRFN-1 and corresponding griffithsin fragment structures.

(A) residues 18–35 of griffithsin in blue (PDB 2GTY), (B) structure of GRFN-1 in red and their overlay (C). Structure of GRFN-1 was obtained from molecular dynamics simulation in water for 50 ns.

Figure 13. Monomeric components of N-linked glycans.

All depicted carbohydrates were used in SPR competition studies. Critical hydroxymethyl moiety is in red and methyl group in position 5 of fucose is in blue.