Development of Immune Modulators Targeting Autoimmune Disorders and Pulmonary Injury: Design, Synthesis, and Biological Evaluation of Sphingolipid-Derived Sphingosine-1 Phosphate Receptor 1 Agonists

Abstract

Multiple sclerosis and asthma account to the most prevalent autoimmune diseases and pose major challenges in modern medicine. The lysophospholipid–activated G protein–coupled receptor family of sphingosine-1-phosphate receptors are central regulators of the eukaryotic native as well as adaptive immune system and are heavily involved in the pathogenesis of respective autoimmune disorders. Therefore, their sphingolipid substrates have been a key target for immunomodulation ever since their function’s discovery. Despite extensive research, a synthetic sphingoid derivative – fingolimod – that acts as a competitive receptor expression level downregulator, featuring prolonged prodrug character and undesired receptor promiscuity, remains the prevalent oral treatment for severe autoimmune disorders since its introduction in 1998. The design, synthesis and medical application of a more bioavailable and synthetically accessible small molecule agent with significantly enhanced receptor specificity is therefore of great medicinal interest.Multiple novel sphingolipid–derived analogs of fingolimod have been designed, analyzed in silico, synthesized, and evaluated for their bioactivity. The lead compound is a fingolimod derivative – tysiponate – which avoids the drawbacks of prodrug nature and low cellular stability and bioavailability the conventional drug on the market features. The compound has been synthesized in a conventional, linear fashion, as well as in a novel, racemic yet significantly more atom-economical synthetic route which exploits photoredox chemistry recently described by Cresswell and coworkers. A purification technique for the target compound in its salt form has been established, allowing the separation of the product of interest from other so far inevitable yet difficult to conventionally separable byproducts. In the pursuit for a both synthetically feasible and more receptor specific autoimmune modulator, computational chemistry utilizing in silico molecule design and analysis of prospective drug candidates regarding their affinity to the target receptor has been performed. An array of potential sphingolipid analogs has been designed and evaluated for their binding affinity to the sphingosine-1-phosphate type 1 receptor, which also poses the target for fingolimod; in silico docking simulations of the substrates to the receptor’s available crystal structure, calculating the potential energy values of the conformers residing and interacting with the active site of the pro- tein, gave insight into what functional moieties contribute to positive binding and possible agonism and which drug candidates were worth the synthetic pursuit. With the findings from the computational experiments and analyses, a novel and quick synthesis of a selection of promising small molecule drug candidates has been developed and executed. The overall strategy described follows a “meta drug” approach, as termed in the laboratory of Dr. Robin Polt at The University of Arizona; as the synthesis of fully functionalized all–carbon systems has proven to be potentially arduous, well established peptide coupling techniques have been used to generate a larger amount of amide–linked analogs in a relatively short amount of time. These obtained “meta drugs” feature very similar bioactive characteristics as their all–carbon counterparts yet are significantly more facile to access synthetically and therefore pose useful candidates for initial in vitro screenings. The compounds synthesized then have been subjected to electric cell–substrate impedance sensing experiments as agonism on the target receptor causes cellular barrier enhancement, resulting in quantitative measurability of the analogs’ potency. Those meta drugs that displayed promising effects in vitro have been then used for further derivatization to optimize both their receptor affinity as well as specificity. Due to their cellular barrier enhancement characteristic, these drug candidates also can be of interest for treatment of acute lung injury and pulmonary endothelial cellular leakage.