Design, Synthesis, and Evaluation of Covalent CADA Analogues as Potent TLR4 Downmodulator for the Treatment of Opioid Use Disorder
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Authors
Oyesakin, Yetunde Mercy
Issue Date
2024
Type
Dissertation
Language
en_US
Keywords
CADA , Cell Densities , Mechanism , Signal Peptides , Synthesis , TLR4
Alternative Title
Abstract
Toll-Like Receptor 4 (TLR4) is involved in the innate immune response and the pathogenesis of opioid use disorder (OUD). Opioids interact not only with neuronal sites but also with glial cells thereby activating these cells, thus leading to the release of neuroexcitatory substances and neuroinflammatory cytokines, contributing to neuroinflammation, pathological pain and neuronal damage. Current therapeutic strategies, though effective, present significant limitations. Given these challenges, alternative therapeutic approaches are essential. Cyclotriazadodecane (CADA) a small molecule initially known to reduce human CD4 (hCD4) protein in a signal peptide (SP) dependent way can downregulate other proteins going through the Sec61 pathway, including TLR4. This work aims to design and synthesize new covalent CADA analogues targeting the SP of TLR4 to address OUD. This study explores the synthesis of different covalent CADA analogues targeting the SP of TLR4, which contains a cysteine residue in its hydrophobic region, making it a viable target for covalent modulation. So far, more than twenty covalent CADA analogues have been synthesized by modifying the tail region and post modification of the sidearm. The synthesis of some of the analogues involves the debenzylation of CADA and subsequent amide coupling reactions to introduce various electrophilic groups to enhance the binding affinity and specificity towards TLR4.The synthesized analogues were characterized using various spectroscopic techniques and confirmed to be pure by elemental analysis. Once the first aim has been accomplished, a novel screening method was developed to evaluate the efficacy of the synthesized CADA analogues in downmodulating TLR4. The assay measured SEAP activity as an indicator of TLR4 activation, providing a quantitative assessment of the downmodulating effects of the CADA analogues. Here in, ten CADA analogues have been screened. While some of them are either toxic or not active, lycorine showed significant TLR4 downmodulation with decreased production of proinflammatory cytokines, suggesting a potential therapeutic effect. This screening method developed provides a robust platform for identifying potent TLR4 downmodulators. Future research should focus on optimizing the pharmacokinetic properties of these CADA analogues and conducting in vivo studies to validate their efficacy and safety. Additionally, exploring the broader applicability of this approach to other neuroinflammatory conditions could further enhance the therapeutic potential of CADA analogues. Oxidative heterofunctionalization of alkenes is a versatile strategy for the synthesis of complex, saturated heterocycles. Additionally, this transformation allows for the formation of medicinally valuable bicyclic compounds via pendant nucleophiles. In this work, we applied previously reported oxidative photocatalytic conditions by the Yoon group to cyclize novel substrates containing N-tosyl amines into medicinally relevant bicyclic scaffolds. Proof of concept for the application of these conditions was established through the isolation of the [3.2.1] bicyclic product. The initial result was optimized utilizing photochemical high throughput experimentation platforms focusing on photocatalyst, solvent, Cu (II), and light sources as variables. The overall yield of this cyclization reaction was improved while also confirming the formation of an additional cyclized side product. We envision further optimization of yield and selectivity as well as substrate scope expansion, which will enable access to other bicyclic cores, including [2.2.1] and [1.1.1] scaffolds.