Development of novel small molecule Toll-like receptors 7 and 8 modulators for cancer immunotherapy

Intracellular targets of the innate immune system that can be modulated by small molecule agents, which offer numerous advantages compared to large biopharmaceuticals, represent big opportunities in pharmacy and innovative possibility to reverse the immune tolerance in the treatment of cancer patients. One of the most important aforementioned targets are Toll-like receptors (TLR).TLR are pattern-recognition receptors that are widely and selectively expressed in multiple cell types including immune cells where they serve as sensors in immune surveillance. This feature has opened the door for TLRs in clinical development of new vaccines and cancer immunotherapeutics. Endosomal TLR, especially type 7 and 8, are prominent targets in cancer drug discovery. TLR7 agonist imiquimod has been used for the treatment of warts and skin malignancies. However, it has only been used locally on the skin due to unfavorable pharmacokinetic properties that induce strong local and systemic inflammatory reactions. Furthermore, many TLR agonists in clinical studies either lack efficacy or cause serious side effects. The aforementioned problematic could be overcome by development of a novel chemical class of small molecule immunomodulators targeting TLR7 or TLR8 that could solve the lack of efficacy and eliminate safety and toxicity issues. Thus, the main objective of this project is to develop small molecule TLR7/8 modulators with new chemical scaffolds incorporating drug-like fragments and functional groups to increase potency and provide favorable safety profile. Iterative drug discovery process, consisting of design, synthesis and biological evaluation will be used and all objectives will be achieved by 4 interconnected Work Packages (WPs). The compounds of interest will be developed from our recently discovered in-house compounds with TLR7 and/or TLR8 activity. Chemistry-driven optimization along with advanced computational drug design approaches (WP1) will lead to more potent compounds with novel chemical scaffolds. Furthermore, all incorporated changes (e.g. functional groups, bioisosteric replacements) will be filtered to avoid the overall chemical reactivity/toxicity and false positive compounds. Analogs of those compounds will be prepared by chemical synthesis incorporating modern synthetic approaches (WP1). TLR7/8 modulatory activity will be then evaluated in a HEK-BlueTMhTLR7(8) reporter assay. Cytotoxicity, immunomodulatory and anticancer properties of selected 3-5 best compounds will be determined in cell-based functional assays (WP2). Measurement of cytokine secretion and dendritic cell experiments (phenotype and function) will test the capacity of TLR7/8 modulators to modify cytokine production and T cells proliferation, which is imperative for effective immune response. The tests will clearly define the extent, as well as quality of developed TLR7/8 modulators, in terms of their capacity to cause induction of effector cells that support either cellular or humoral (antibody-mediated) immunity. For the most promising TLR7/8 modulators from functional assays in vivo antitumor activity will be determined in a mouse colorectal tumor model (WP3). The suppression of tumor growth in CT26 tumor-bearing mice will be tested. Finally, the research activities will be carefully managed and coordinated by PI and WP4 during the entire duration of the project. The results from research will be disseminated and made available to the scientific community. To sum up, our designed, synthesized and optimized compounds with novel chemical scaffolds acting on key immunomodulatory targets, i.e. TLRs, will provide an important starting point in the pursuit of a lead compound or a potential clinical candidate. The project's research will be published in high-ranking journals and patent applications. The research achievements and expertise of PI and other project members and availability of all required equipment demonstrate the feasibility of this project.