Saturday, May 18, 2024
5/18/2024
Project Summary. New anti-inflammatory therapies are needed to help the millions of Americans that are afflicted by one or more chronic inflammatory conditions. Conventional anti-inflammatory drugs are ineffective and limited by deleterious side-effects that result from administering drugs systemically and at high doses. Many chronic inflammatory conditions manifest locally, not systemically. Localized delivery of natural immunoregulatory molecules is an attractive strategy to treat chronic local inflammation. The proposed research will develop a new anti-inflammatory therapy based on synthetic multivalent assemblies of galectin-1 (“G1”) and galectin-3 (“G3”). G1 and G3 are natural regulators of inflammation that signal changes in immune cell behavior via membrane glycan recognition. Despite some reported successes of wild-type G1 and G3 as anti- inflammatory therapies, their potency is ultimately limited by their low carbohydrate-binding affinity and their unstable active quaternary structures. To address these limitations, our approach joins G1 and G3 into synthetic multivalent nanoassemblies. We make these nanoassemblies through recombinant fusion of G1 and G3 onto the N- and C-termini of peptide linkers that form a-helical coiled-coils. Using this approach, we created G1/G3 Zipper, a heterotetramer with two G1 and two G3 domains (i.e., a dimer-of-dimers). G1/G3 Zipper was shown to have significantly greater signal potency than wild-type G1 and G3 in vitro, and acts through different receptors and different pathways than the wild-type proteins. Preliminary data demonstrate that G1/G3 Zipper can suppress sterile inflammation induced by l-Carrageenan, whereas an engineered G1 homodimer is ineffective. G1/G3 Zipper can also resolve imiquimod-induced psoriasis. Using a similar coiled-coil scaffold approach, we recently demonstrated that the signaling activity of G3 increases as the number of G3 domains increases from two to six. Informed by these data, the guiding hypothesis of the proposed research is that the anti- inflammatory potency of G1/G3 Zipper constructs can be maximized by increasing the number of G1 and G3 domains. To test this hypothesis, Specific Aim 1 is to measure the signaling activity of G1/G3 Zipper constructs with four or six G1 and G3 domains using various cell models of G1 and G3 signaling. Specific Aim 2 is to evaluate the effectiveness of G1/G3 Zipper constructs to suppress local inflammation using the l-Carrageenan and imiquimod-induced psoriasis models. We will use ELISA, bulk RNAseq, and in vivo imaging to identify the mechanisms of action of G1/G3 Zipper. We will use in vivo imaging and histology to establish G1/G3 Zipper pharmacokinetics. We will use humoral immunity models to assess G1/G3 Zipper safety. Completion of the proposed studies will establish our innovative G1/G3 Zipper construct as a new class of anti-inflammatory therapy. Harnessing the immunomodulatory activity of G1 and G3 to treat chronic local inflammation has the potential to impact nearly all aspects of human health. Establishing valency-function relationships of G1/G3 Zipper in two inflammation models will be valuable for translation of galectin therapies.
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