Biological evaluation of novel therapeutics for treatment of celiac sprue.

摘要

Celiac sprue is an inheritable inflammatory disease of the upper small intestine found in up to 0.5--1% of the population in certain parts of the world. Investigations into the molecular basis of celiac sprue have unmasked some of the key proteins involved in disease pathogenesis, including the cereal storage protein gluten, the deamidating enzyme transglutaminase 2, and the disease-associated major histocompatibility complex protein HLA-DQ2. Therapeutic strategies targeting each of these disease-relevant proteins have been developed. However, the absence of an animal model for celiac sprue prohibits direct in vivo evaluation of the therapeutic efficacy of these novel drug candidates. Therefore, alternative disease-relevant biological systems are necessary for pre-clinical development and lead candidate identification of such therapeutics.;This thesis describes the development and utilization of disease-relevant biological systems to test the therapeutic potential of (1) supplemental proteases that enhance gluten digestion; (2) small molecule transglutaminase 2 inhibitors, and (3) gluten-peptide-based blockers of HLA-DQ2. Using small intestinal biopsy-derived T cells from celiac sprue patients, a cysteine endoprotease from barley, EP-B2, was shown to significantly reduce the number of inflammatory T cell gluten epitopes surviving simulated gastric digestion of gluten. Synergism between EP-B2 and prolyl endopeptidases (PEPs) from Flavobacterium meningosepticum (FM) and Sphingomonas capsulate (SC) suggested that a combination enzyme therapy comprised of EP-B2 supplemented with either FM PEP or SC PEP was an especially potent therapeutic. Cell culture and in vivo experiments using active-site directed irreversible inhibitors of the deamidating enzyme transglutaminase 2 showed that the majority of this enzyme is latent under normal physiological conditions. However, transglutaminase 2 could be activated both in cell culture and in vivo following wounding. Transglutaminase 2 inhibitors were shown to effectively block activated transglutaminase 2 in cell culture. Finally, gluten peptide-based blockers of HLA-DQ2 with chemically modified solvent-exposed amino acid side chains were designed to reduce antigen presentation of gluten peptides by binding HLA-DQ2 without causing T cell proliferation. By using B-lymphoblastoid cells as model antigen presenting cells, two HLA-DQ2 blockers were shown to reduce the T cell proliferation caused by a highly inflammatory gluten peptide and digested gluten.