Oxysterols are oxygenated metabolites of cholesterol formed in the human body and are involved in a plethora of physiological and pathological processes such as lipid homeostasis, inflammation, innate and adaptive immunity, cancer, and brain degenerative diseases. Specifically, 25-hydroxycholesterol (25HC) is now established as an important regulator of the immune system, and is produced by immune cells in response to viral infection and activation of pattern recognition receptors. Recently, we uncovered a novel cellular mechanism of 25HC-mediated regulation of the proinflammatory response. We showed that 25HC amplifies the activation of immune cells and increases the production of immune mediators such as TNF and IL-6, by directly binding to ?v?3 and ?5?1 integrins and activating the integrin-focal adhesion kinase pathway. We also discovered that 25HC binds to integrins at a novel binding site (site 2), distinct from the site where the extracellular matrix (ECM) ligands containing an Arg-Gly-ASP (RGD) motif are known to bind. Binding of 25HC at site 2 produces significant conformational changes in the specificity-determining loop (SDL) of integrins, near the RGD-binding site. The effect of such conformational changes in the SDL on the binding of ECM ligands, as well as the basis of 25HC- mediated allosteric signaling mechanism underlying integrin activation, are not known. Our hypothesis is that binding of 25HC to integrins at site 2 triggers conformational changes in the SDL that result in efficient binding of ECM ligands producing further modification of innate inflammatory response. We also hypothesize that small molecule modulators blocking 25HC-integrin interaction would serve as an efficient anti-inflammatory therapeutic strategy to combat various inflammatory diseases. Accordingly, the central objective of this proposal is to elucidate the molecular mechanisms of integrin activation by oxysterols and to identify selective small molecule modulators targeting site 2 of integrins for potential therapeutic applications. The objective of this project will be accomplished by the following three specific aims: 1) elucidate the molecular basis and conformational dynamics of integrin activation by 25HC; 2) examine the molecular recognition of integrins by non-25HC oxysterols; and 3) identify and evaluate small-molecule modulators targeting the 25HC binding site of integrins. We will utilize state-of-the-art computational techniques such as molecular docking, molecular dynamics simulations, and pharmacophore-based virtual screening to delineate the structural basis and conformational dynamics involved in activation of integrins and to identify high affinity ligands for site 2 of integrins. In addition, our well-established in vitro and in vivo models will be employed to validate our in silico findings and evaluate top ligands. Our multidisciplinary approach is innovative and together, the proposed studies will have a broad impact by offering fundamental insights to the interplay between oxysterols and integrins that culminates in amplification of the inflammatory response. Furthermore, this project will identify one or more modulators of integrin-25HC interactions, thereby advancing potential anti-inflammatory therapies for immunologic and infectious diseases.
The proposed research is relevant to public health, because the oxysterols and integrins are involved in many pathological conditions including autoimmune disorders, cancer, inflammation, cardiovascular, and brain diseases. This study will offer fundamental insights into the molecular basis of oxysterols-mediated activation of integrins and will enable efforts toward the development of potential small-molecule therapeutics. Thus, this proposal is relevant to the NIH?s mission to seek fundamental knowledge to help prevent and treat immunologic and infectious diseases.
