Interleukin-1 Receptor Modulators: Drawing Links between Structures, Biased Signaling, and in vivo Efficacy in Models of Preterm Birth and Retinopathy of Prematurity

• Main Author: Cheng, Colin W. H
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2019
• Subjects: Amniotic fluid; Brain research; Cytokines; Immunology; Molecular biology; Obstetrics; Ophthalmology; Pediatrics; Peptides; Pharmacology; Premature birth; Proteins
• ISBN: 9798597049410

Introduction: Preterm birth (PTB) and subsequently, retinopathy of prematurity (ROP), are two neonatal pathologies associated with a dysregulated inflammatory response. Interleukin (IL)-1 is strongly implicated in both conditions; however, current therapies against IL-1 are large molecules and indiscriminately block all IL-1 signaling pathways, causing undesirable immunosuppressive effects. We previously developed an allosteric modulator of the IL-1 receptor (all-D peptide rytvela, hereafter referred to as 101.10) that was effective in several animal models of inflammatory diseases in an NF-κB-independent manner. To further our understanding of 101.10’s structural-activity relationships and to elucidate the roles of the various signaling pathways in PTB and ROP, we synthesised a panel of twelve 101.10 lactam derivatives with varying chiralities in residues 3 and 4 and tested them in vitro on cell lines, and in vivo in PTB and ROP models. Methods: RAW-blue or HEK-blue cells were stimulated with IL-1β after pre-treatment with our derivatives. The QUANTI-blue spectroscopic assay was used to quantify secreted alkaline phosphatase, a reporter gene of NF-κB activity. Western blots were used to quantify phosphorylation of ROCK2, p38 and JNK. Quantiative PCR was also used to determine the expression of pro-inflammatory genes in the cells. The ability of our derivatives to displace 101.10 from the IL-1 receptor was assessed with radioligand binding assays. Derivatives were then tested in vivo in a CD-1 mouse model of LPS-induced PTB, and a Sprague Dawley rat model of ROP induced by exposure to 80% O2. Results: All derivatives did not inhibit NF-kB signaling, but most inhibited ROCK2 phosphorylation. Derivatives with an L-valine were stronger inhibitors of p38 phosphorylation and weaker inhibitors of JNK phosphorylation than those with a D-valine. Notably, D-valine derivatives were stronger inhibitors of PTB. The efficacy of D-valine derivatives in vivo PTB did not completely translate into efficacy in ROP. Nonetheless, it was observed that both JNK and ROCK2 inhibition were necessary for ROP prevention, while JNK alone prevented PTB. Conclusions: Selective modulation of IL-1 signaling, especially JNK and ROCK2 phosphorylation, without affecting NF-κB is a feasible strategy for preventing PTB and ROP. Our small molecules could offer advantages over existing therapies, such as reduced side effects and easier administration.