Project 2: Why are mucins so gigantic and is it safe/effective to sever them therapeutically?

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Project 2, “Why are mucins so gigantic and is it safe/effective to sever them therapeutically?”, Richard C. Boucher, MD, PI, proposes to study fundamental questions with respect to mucus transport in health and how to therapeutically restore mucus clearance in muco-obstructed patients. The project first focuses on a simple question, i.e., why are such gigantic mucin multimers (~ 120 monomers, 300 MDa, 250 nm radius of gyration, Rg) synthesized and secreted by normal airway epithelia? We hypothesize that large multimers are the most economical way to generate the mucus gel-like properties needed for transport. Studies will focus on mucin molecular weight (MW), molecular size (radius of gyration, Rg), concentration (c), overlap concentrations (c*), gel-dependent biophysical properties, and transportability. A corollary goal is to test the hypothesis that mucolytic agents, i.e., agents that decrease multimer length/size, are attractive therapeutic agents for muco-obstructive lung disease. Our approach to investigate both hypotheses rests on the successful generation of a CRISPR knockout mutant mouse (Muc5b D3 cysteine 1128-alanine, cysteine 1170-alanine) that selectively synthesizes and secretes mucin dimers (MW ~ 4 MDa, Rg = 40 nm). SA1 will test the hypothesis that multimeric mucins (large multimers) efficiently occupy space/volume in the mucus layer and, consequently, generate the overlap conditions (c*) needed to produce the gel-like mucus properties required for transport. Structural features of wild- type (WT) vs Muc5b dimeric mucins, utilizing light scattering, neutron scattering, and EM technologies, juxtaposed to biophysical properties, e.g., viscoelastic properties, osmotic moduli, cohesion, and adhesion, will be measured and related to cilial and cough-dependent transport in vitro. SA1 will also test the specific hypothesis that dimeric mucins, because of their small Rg, will not reach the c* conditions under basal conditions (secretory rates) required to generate the elastic properties of a gel needed for cilial-dependent mucus clearance. SA2 will then test the hypothesis that a mucus comprised of small MW mucins is not transportable by cilia in vivo and mucus accumulation in the respiratory tract will result. Responses of WT vs dimeric mice to provocations that perturb mucin concentrations will be tested to explore the requirement of multimeric mucins for host defense. SA3 will test the hypothesis that reducing agents aid mucus clearance in disease but not health. Studies will include comparisons of actions of a novel, efficient thiol-based therapeutic agent (P2165) on WT vs dimeric mucins in vitro and mice in vivo. The goal is to search for potential favorable effects of reducing multimer length on the mucus viscosity required for cough clearance and identify off-target effects mediated by reduction of other intra-mucin cysteines that may produce untoward mucin aggregation/stickiness. The deliverables of the project are to: 1) quantitate the relationships between mucin multimer size, space-occupying characteristics (c*), and mucus function in health; and 2) characterize the mucolytic agent therapeutic index with respect to on- vs off- target effects and provide a roadmap for development of novel mucolytic therapeutics for patients in need.