Research Projects

Based on the detailed understanding of the Structure Activity Relationship studies, new desmuramylpeptides that activate NOD2 will be designed. The designed molecules will be synthesized using solid-phase peptide synthesis (SPPS) as well as classic liquid-phase peptide synthesis. The specific immune cell activation via synthetic NOD2 ligands will be studied by analyzing the induction of TNF-α and IL-6 in PBMCs. The immune response generated by the vaccine formulations, including antibody production, T-cell activation, and cytokine production will be investigated. The contemplated heterocycles can be synthesized following established coupling pathways and will be used to furnish influential derivatives of desmuramylpeptides. The synthesized compounds will also be evaluated following the literature protocols to check the cytotoxicity toward HEK-Blue. HEK-Blue NOD2 cells will be treated with desmuramylpeptides, alongside MDP as a reference compound, for more than 24 hours at concentrations of 2 or 20 μM. HEK-Blue NOD2 cells will first be preincubated with a NOD2 antagonist (10 μM) before being stimulated with synthesized desmuramylpeptides or MDP (2 μM) for over 24 hours. Following this incubation period, SEAP activity will be measured in the supernatant. The observed stimulation by desmuramylpeptides and MDP (2 μM) will be normalized against control cells to confirm that the induced NF-κB activity resulted specifically from the NOD2 agonistic properties of these compounds. Cytokine production will be measured through the BD Cytometric Bead Array (CBA) Human Th1/Th2/Th17 Kit, and flow cytometry will be performed using a FACS flow cytometer. Standard curves will be created using recombinant cytokines provided in the kit. The established docking framework will be utilized to design novel analogs and develop a comprehensive Structure-Activity Relationship (SAR). Furthermore, molecular dynamics simulations will be performed to assess the stability of the NOD2-Ligand complexes.