New nanostructures inhibiting human mannose binding lectin identified by a novel surface plasmon resonance assay

Circulating mannose-binding lectin (MBL) plays an important role in the progression of tissue damage caused by ischemic events, an account of its high-affinity, multivalent binding to carbohydrate arrays exposed on damaged endothelium (damage-associated molecular patterns, DAMPS). MBL inhibitors have therefore been proposed as a novel protective therapeutic strategy to prevent secondary injury. We developed a new, convenient, robust surface plasmon resonance (SPR) assay for in vitro screening of compounds interfering with the binding of native human MBL to an appropriate chip surface functionalized with a pattern of sugar moieties mimicking DAMPS. We also characterized the procedure to regenerate the chip surface after each experimental session, accomplished by sequential cleaning with piranha solution followed by UV exposure. The SPR assay detects the specific binding of human recombinant MBL and native MBL present in human serum, and can identify inhibitors of this binding. We observed inhibitory effects of mannose (IC50 ≅ 5 mM), of a nine mannose residues carrying glycan (IC50 ≅ 0.33 mg/mL, corresponding to ~ 175 μM), and mainly mannose-coated gold nanoparticles (IC50 ≅ 1.1 μg /mL). These in vitro results serve as a basis for testing the protective properties of these molecules/nanoparticles later inthe more expensive and time-consuming studies in cells and animal models of MBL mediated-injuries. [Display omitted] This surface plasmon resonance (SPR) assay was developed to recreate the natural condition in which circulating mannose binding lectin (MBL) binds the sugar moieties exposed on cell membranes after an injury (damage associated molecular patterns, DAMPs). In this novel assay MBL − with or without inhibitors − flows over and binds a suitable carbohydrate pattern exposed on a SPR sensor chip. •Mannose binding lectin (MBL) inhibitors may protect from secondary ischemic injury.•A unique SPR-based assay was developed for a convenient screening of MBL inhibitors.•Novel multivalent mannosylated nanoconstructs were developed and characterized.•These nanoconstructs inhibited MBL binding to a mannose-exposing sensor chip.•The new methodological solutions and tools can be applied in diverse research areas.