Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Aug 27;15(37):15352-15366.
doi: 10.1039/d4sc02980a. Online ahead of print.

Unprecedented selectivity for homologous lectin targets: differential targeting of the viral receptors L-SIGN and DC-SIGN

Clara Delaunay  1 , Sara Pollastri  2 , Michel Thépaut  1 , Gianluca Cavazzoli  2 , Laura Belvisi  2 , Clémentine Bouchikri  1 , Nuria Labiod  3 , Fatima Lasala  3 , Ana Gimeno  4 , Antonio Franconetti  4 , Jesús Jiménez-Barbero  4   5   6 , Ana Ardá  4   5 , Rafael Delgado  3   7 , Anna Bernardi  2 , Franck Fieschi  1   8
Affiliations

Unprecedented selectivity for homologous lectin targets: differential targeting of the viral receptors L-SIGN and DC-SIGN

Clara Delaunay et al. Chem Sci. .

Abstract

DC-SIGN (CD209) and L-SIGN (CD209L) are two C-type lectin receptors (CLRs) that facilitate SARS-CoV-2 infections as viral co-receptors. SARS-CoV-2 manipulates both DC-SIGN and L-SIGN for enhanced infection, leading to interest in developing receptor antagonists. Despite their structural similarity (82% sequence identity), they function differently. DC-SIGN, found in dendritic cells, shapes the immune response by recognizing pathogen-associated carbohydrate patterns. In contrast, L-SIGN, expressed in airway epithelial endothelial cells, is not directly involved in immunity. COVID-19's primary threat is the hyperactivation of the immune system, potentially reinforced if DC-SIGN engages with exogenous ligands. Therefore, L-SIGN, co-localized with ACE2-expressing cells in the respiratory tract, is a more suitable target for anti-adhesion therapy. However, designing a selective ligand for L-SIGN is challenging due to the high sequence identity of the Carbohydrate Recognition Domains (CRDs) of the two lectins. We here present Man84, a mannose ring modified with a methylene guanidine triazole at position 2. It binds L-SIGN with a K D of 12.7μM ± 1 μM (ITC) and is the first known L-SIGN selective ligand, showing 50-fold selectivity over DC-SIGN (SPR). The X-ray structure of the L-SIGN CRD/Man84 complex reveals the guanidinium group's role in achieving steric and electrostatic complementarity with L-SIGN. This allows us to trace the source of selectivity to a single amino acid difference between the two CRDs. NMR analysis confirms the binding mode in solution, highlighting Man84's conformational selection upon complex formation. Dimeric versions of Man84 achieve additional selectivity and avidity in the low nanomolar range. These compounds selectively inhibit L-SIGN dependent trans-infection by SARS-CoV-2 and Ebola virus. Man84 and its dimeric constructs display the best affinity and avidity reported to date for low-valency glycomimetics targeting CLRs. They are promising tools for competing with SARS-CoV-2 anchoring in the respiratory tract and have potential for other medical applications.

PubMed Disclaimer

Conflict of interest statement

The authors declare the following competing financial interest(s): S. P., C. D., M. T., F. F., and A. B. declare the filing of a patent covering the use of glycomimetic L-SIGN ligands as antagonist, anti-viral adhesion, and for targeting human L-SIGN-expressing cells.

Figures

Fig. 1
Fig. 1. Monovalent ligands analyzed in this study.
Scheme 1
Scheme 1. Synthesis of 2 (Man84).
Scheme 2
Scheme 2. Synthesis of the monovalent ligands 8 and 9.
Scheme 3
Scheme 3. Synthesis of PM68.
Scheme 4
Scheme 4. Synthesis of PM69 was achieved both by direct guanidinylation of PM68 (upper panel), or via CuAAC of 15 with Rod 3 (lower panel), with similar results.
Fig. 2
Fig. 2. The four divalent ligands analyzed in this study: PM68 and PM70 are a divalent presentation of the amino-substituted ligand Man79; PM69 and PM74 are divalent presentations of the guanidine-substituted ligand Man84. For each monovalent spearhead, a short linker and a long linker presentation were prepared.
Fig. 3
Fig. 3. (A) Sensorgrams of DC-SIGN binding inhibition (left panel) and L-SIGN binding inhibition (right panel) by Man84. Range of Man84 concentrations goes from 5 mM to 10 μM by serial dilution by a factor of 2 with same color code for both DC-SIGN and L-SIGN inhibition. (B) SPR inhibition curves for Man79 (circle) and Man84 (square) (see ESI for all sensorgrams). Inhibition curves concerning DC-SIGN are represented in blue and L-SIGN in orange. Man79 data, were already described in Pollastri et al., and are shown here for direct comparison with Man84 data. (C) IC50 values of Man79 and Man84 for DC-SIGN (in blue) and L-SIGN (in orange). Values represented are the corresponding IC50.
Fig. 4
Fig. 4. Titration of the Man84 ligand at 2 mM to L-SIGN ECD (172 μM) by ITC. (A) Representative data among a series of 3 of the titration thermograms obtained (see ESI for all ITC titration experiments). (B) Data integration with fitted curve, using 1 : 1 binding model. (C) Average thermodynamic parameters values obtained following the L-SIGN CRD/Man84 complex formation.
Fig. 5
Fig. 5. 3D structural binding mode of Man84 within L-SIGN CRD and mechanism of selectivity. (A) Structure of the L-SIGN CRD/Man84 complex (PDB: 8RCY). Man84 is shown superimposed over the Fo − Fc electron density map (light blue, 2σ contour) within L-SIGN CRD. Side chain of residues involved in the binding are represented as sticks. H-Bonds are represented as yellow dashed lines, Ca2+ coordination bond as magenta dashed line and π-cation interaction by green dashed lines. Water molecules are represented as red spheres. (B) Alignment with the CRD of DC-SIGN (PDB: 2IT6) for comparative purposes. Side chain of corresponding residue from DC-SIGN CRD are presented as cyan sticks and labelled. Electrostatic surfaces of L-SIGN CRD (C) and DC-SIGN CRD (D) were calculated via the PyMol software, with complexed Man84 represented in yellow in L-SIGN CRD or in grey in DC-SIGN CRD where it has been added by structural alignment for comparison. The Ca2+ ion in the binding site is represented by a grey sphere (see Table S1 for data collection and structure refinement statistics).
Fig. 6
Fig. 6. The binding of Man84 to L-SIGN (top) and DC-SIGN (bottom) in solution, by monitoring NMR signals of the corresponding protein in the absence and presence of the ligand (30 equivalents in the case of L-SIGN, and 100 equivalents for DC-SIGN). On the left, plots for the CSP analysis (average chemical shift difference between protein free and bound states) and on the right 3D cartoon representation of the corresponding protein highlighting the most affected residues in the CSP analysis: in red residues with CSP above twice the standard deviation (SD) of the whole data set, and orange residues with CSP above the SD. Residues in black are not assigned or are prolines. Residues in light blue disappear in the bound state. The ligands are in yellow and Ca2+ ions in dark blue. For L-SIGN, residues involved in direct intermolecular interactions with Man84 as found by X-ray crystallography are annotated in the CSP plots.
Fig. 7
Fig. 7. The binding of Man84 to DC-SIGN and L-SIGN in solution from the ligand perspective. Blue contours correspond to positive NOE, while black contours correspond to negative NOE. (A) NOESY spectrum of Man84 showing the positive NOE correlations of H_Tz. (B) The same region of the trNOESY spectrum of Man84 in the presence of DC-SIGN (1 : 17 protein : ligand molar ratio). (C) The same region of the trNOESY spectrum of Man84 in the presence of L-SIGN (1 : 10 protein : ligand molar ratio). (D) Structure of Man84 showing the free rotation around the C2(Man)–N(Triazol) bond. (E) The L-SIGN-bound conformation of Man84, as derived by NMR interaction data. This conformation corresponds to the one observed in the X-ray structure (Fig. 5A).
Fig. 8
Fig. 8. SPR interaction tests with titration of PM68 (filled circles), PM69 (empty circles), PM70 (filled squares) and PM74 (empty squares) on DC-SIGN ECD (blue curves) and L-SIGN ECD (orange curves) oriented surfaces. Range of interactions were performed in duplicates.
Fig. 9
Fig. 9. Topology of DC-SIGN (panel A) and L-SIGN (panel B) active sites. Tetrameric representation issued form SAXS, X-ray crystallography studies and molecular modelling from ref. and . Figures made with PyMol.
Fig. 10
Fig. 10. Trans-infection assays of EBOV pseudotyped rVSV-luc in VeroE6 mediated by Jurkat DC-SIGN (left panel) and Jurkat L-SIGN (right panel). Results are presented as percentage of EBOV trans-infection control in the presence of compounds: PM ligands and mannan (Man) as compared to trans-infection of EBOV in the absence of inhibitors. The results were analyzed using GraphPad Prism v8.
Fig. 11
Fig. 11. Trans-infection assays of SARS-CoV-2 pseudotyped rVSV-luc in VeroE6 mediated by Jurkat DC-SIGN (left panel) and Jurkat L-SIGN (right panel). Results are presented as percentage of EBOV trans-infection control in the presence of compounds: PMs and mannan (Man) as compared to trans-infection of EBOV in the absence of inhibitors. The results were analyzed using GraphPad Prism v8.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Takeuchi O. Akira S. Cell. 2010;140:805–820. doi: 10.1016/j.cell.2010.01.022. - DOI - PubMed
    1. Geijtenbeek T. B. H. Gringhuis S. I. Nat. Rev. Immunol. 2009;9:465–479. doi: 10.1038/nri2569. - DOI - PMC - PubMed
    1. Van Vliet S. J. Dunnen J. D. Gringhuis S. I. Geijtenbeek T. B. Van Kooyk Y. Curr. Opin. Immunol. 2007;19:435–440. doi: 10.1016/j.coi.2007.05.006. - DOI - PubMed
    1. Kinberger G. A. Prakash T. P. Yu J. Vasquez G. Low A. Chappell A. Schmidt K. Murray H. M. Gaus H. Swayze E. E. Seth P. P. Bioorg. Med. Chem. Lett. 2016;26:3690–3693. doi: 10.1016/j.bmcl.2016.05.084. - DOI - PubMed
    1. Dorscheid D. R. Conforti A. E. Hamann K. J. Rabe K. F. White S. R. Histochem. J. 1999;31:145–151. doi: 10.1023/A:1003599916558. - DOI - PubMed

LinkOut - more resources