In silico-aided design of a glycan ligand of sialoadhesin for in vivo targeting of macrophages

Abstract

Cell-specific delivery of therapeutic agents using ligand targeting is gaining interest because of its potential for increased efficacy and reduced side effects. The challenge is to develop a suitable ligand for a cell-surface receptor that is selectively expressed on the desired cell. Sialoadhesin (Sn, Siglec-1, CD169), a sialic acid-binding immunoglobulin-like lectin (Siglec) expressed on subsets of resident and inflammatory macrophages, is an attractive target for the development of a ligand-targeted delivery system. Here we report the development of a high-affinity and selective ligand for Sn that is an analogue of the natural ligand and is capable of targeting liposomal nanoparticles to Sn-expressing cells in vivo. An efficient in silico screen of a library of ∼8400 carboxylic acids was the key to identifying novel 9-N-acyl-substituted N-acetylneuramic acid (Neu5Ac) substituents as potential lead compounds. A small panel of targets were selected from the screen and synthesized to evaluate their affinities and selectivities. The most potent of these Sn ligands, 9-N-(4H-thieno[3,2-c]chromene-2-carbamoyl)-Neu5Acα2-3Galβ1-4GlcNAc ((TCC)Neu5Ac), was conjugated to lipids for display on a liposomal nanoparticle for evaluation of targeted delivery to cells. The (TCC)Neu5Ac liposomes were found to target liposomes selectively to cells expressing either murine or human Sn in vitro, and when administered to mice, they exhibited in vivo targeting to Sn-positive macrophages.

Figure 1

Representative structures from the in silico screening strategy to identify high affinity ligands of Sialoadhesin (Sn). (A) Representative carboxylic acid from a commercial building block library that were screened as potential substituents of 9-NH₂-Neu5Ac. (B-E) Four representative solutions from the tethered docking approach highlighting varying docking poses of the tethered acid substituent. Conformations of the respective substituents were calculated and conjugated via in silico amide coupling to 9-NH₂-Neu5Ac, which was fixed within the binding site. The top 3000 hits from this initial docking evaluation were further inspected using AutoDock. (F) Representative AutoDock solution for the selected carboxylic acid coupled to 9-NH₂-Neu5Ac. Oxygen, carbon, and nitrogen atoms are highlighted in red, white, and blue, respectively.

Figure 2

Inhibitory potencies of selected sialoside analogs in a murine Sn competitive bead binding assay. Each compound was analyzed in triplicate for inhibition of the binding of mSn-Fc chimera to Neu5Acα2-3-Galβ1-4GlcNAc coated magnetic beads as described in the Supporting information. See Table 1 for glycans 1, 2 and 4.

Figure 3

Fluorescence assisted cell sorting (FACS) analysis for in vitro binding of targeted ^TCCNeu5Ac or non-targeted “naked” liposomes to cells expressing murine and human Siglecs. Binding is shown as mean fluorescence intensity (MFI) ±SD (n = 3).

Figure 4

^TCCNeu5Ac liposomes target Sn+ macrophages in vivo. A) Wild type mice were injected subcutaneously with fluorescently labeled naked or ^TCCNeu5Ac liposomes and the neighboring lymph nodes were harvested after 2.5 h. Cells were stained with various antibodies as described in the Supporting Information and analyzed by flow cytometry. B) Wild type and Sn KO mice (n = 3 in each group) were injected with the liposomes. The binding of liposomes to macrophages (TCRb⁻NK1.1⁻CD19⁻CD11b⁺) were analyzed by flow cytometry. The geometric mean of fluorescent intensity (MFI) were plotted.

Scheme 1. Chemo-enzymatic synthesis of C-9 N-acyl modified sialoside analogues.^a

^aReagents and conditions: a) CMP-9-NH₂-Neu5Ac, PmST1; b) RC(O)NHS, THF, H₂O (1-9, see Table 1 for R).