Principal Investigator: Gerardo R. Vasta, Ph.D., Professor
Grant Title: Galectin Inhibitors for Sepsis Prevention and Therapy
Abstract
1. Objectives: Our prior studies showed that galectins expressed and secreted by the airway
epithelia contribute to the enhanced susceptibility of influenza patients to pneumococcal
pneumonia and ensuing hypercytokinemia. Specifically, we showed that unmasking of galactosyl
moieties by the influenza A virus neuraminidase on the airway epithelial surface that takes place
upon a primary viral infection, leads to enhanced galectin binding and pneumococcal adhesion.
Thus, we proposed that inhibiting the binding of galectins to the airway epithelia during IVA
infection will prevent or ameliorate the pneumococcal superinfection and ensuing sepsis. To test
this hypothesis, we conducted studies with the following objectives: (a) identify and characterize
galectin receptors on the airway cell surface, and the effectiveness of novel high affinity/avidity
multivalent inhibitors of galectin binding (MSIGB) in disrupting galectin-receptor interactions,and (b) assess the effectiveness of MSIGB for preventing a dysregulated cytokine response
2. Methods used: For the identification of galectin receptors on the airway epithelial cell surface,
we used affinity chromatography of A549 cell extracts on immobilized human recombinantgalectin 1 (Gal1) and galectin 3 (Gal3). The eluted galectin ligands were electrophoresed (PAGE),
transferred to PVDF membranes, and developed with recombinant Gal1 and Gal3, and specificantibodies against selected cell surface receptors. The in vitro characterization of the specificity of
the galectin-receptor interactions was assessed by ELISA, testing binding-inhibition with selectedoligosaccharides and glycoproteins. The design, synthesis and validation of MSIGB, was carriedout in collaboration with Dr. L-X. Wang (UMDCP). Well-established methods in Dr. Wang’s lab
were applied for the synthesis, analysis, and spectroscopic validation of the MSIGB that werebased on a cyclodextrin (CD) core. Binding-inhibition specificity of MSIGB was measured byELISA and affinity by surface plasmon resonance (SPR). The effectiveness of MSIGB indisrupting galectin-mediated downregulation of SOCS expression was carried by standard andqRT-PCR. The preventive/therapeutic value of the MSIGB in our mouse model for post-influenza
pneumonia/sepsis is currently assessed in collaboration with Dr. A.S. Cross, UMB).
3. Results: The first group of experiments was focused on the identification of Gal3 receptors on
the airway cell surface and revealed the presence of α3β1 integrin and MUC1; ongoing studies areaimed at confirming the presence of CD147. The synthesis of inhibitors for Gal3 binding wassuccessful in that both monovalent and CD- based multivalent inhibitors were synthesized, with
non-reducing terminal galactosyl moieties, namely lactose (Lac), N-acetylgalactosamine(GalNAc), and the Thompsen-Friedenreich disaccharide (TFD; Galβ1,3GalNAc). The MSIGB
were effective not only in preventing binding of Gal3 to desialylated glycoproteins such as
asialofetuin, but also to airway epithelial cells (A549 cell line). Most importantly, the results
showed that MSIGB are effective in hindering Gal3 binding. Further, our studies demonstrated
that by preventing the binding of Gal3 to the airway cell surface the synthetic inhibitors were alsoeffective in preventing the downregulation of SOCS, a key achievement for the prevention of thecytokine storm that results from the post-influenza pneumococcal pneumonia. Animal testing of
the inhibitors’ efficacy and lack of toxicity is ongoing.