<p>  </p> <p>Cyclic dinucleotides (CDNs) such as the bacterial CDNs (cyclic-di-AMP, cyclic-di-GMP and 3’3’cyclic GMP-AMP) and mammalian CDN, 2’3’-cGAMP, are essential immune response second messenger signaling molecules. These CDNs act via Stimulator of interferon genes (STING)-TANK Binding Kinase 1 (TBK1)-Interferon Regulatory Factor 3 (IRF3) pathway. However, data from our lab and others indicate that beyond the classical STING-TBK1-IRF3 pathway, CDNs also regulate other signaling axes related to both inflammatory and non-inflammatory pathways. But, a global view of how these CDNs affect signaling in diverse cells or through non-STING pathways is lacking. There is also paucity of data on CDN modulated kinases and no global assessment of phosphorylation events that follow cyclic GMP AMP synthase (cGAS)-STING axis stimulation in immune cells. Herein, I have used a proteomics approach to determine signaling pathways regulated by bacterial CDNs, c-di-GMP and c-di-AMP in human gingival fibroblasts such as pathways related to nucleotide excision repair (NER) which ordinarily do not channel through STING (Chapter 3). Additionally, with the use of phosphoproteomics and bioinformatics, this project accomplished a system-wide phosphorylation analyses of T cells treated with 2’3’cGAMP and showed that 2’3’cGAMP impact various, yet unreported critical kinases (E.g. LCK, ZAP70, ARG2) and signaling pathways important for T cell function (Chapter 4). Asides known interferon signaling, these differentially phosphorylated kinases were involved in T cell receptor (TCR) signaling, myeloid cell differentiation, cell cycle regulation, and regulation of double strand break repair. </p> <p>Another area of interest addressed by this project is the discovery of novel antibacterial agents against multi-drug resistant (MDR) bacteria. Thus, in Chapters 5 and 6, I show the identification, antibacterial activity and characterization of <strong>HSD1835</strong> and <strong>HSD1919 </strong>as novel SF5 and SCF-containing membrane active compounds, highly potent against preformed MDR biofilms with fast bactericidal activity against persister bacteria. Plus, an exciting addition to the fight against MDR bacteria in Chapter 7, the discovery of <strong>HSD1624</strong> and analogs, which are able to re-sensitize MDR and colistin resistant bacteria such as <em>Pseudomonas aeruginosa</em> from a colistin MIC of 1024 μg/mL to 0.03 μg/mL (64000-fold reduction). Ultimately, these compounds could be translated into anti-biofilm and, anti-MDR bacteria therapeutics, preventing repeated surgeries due to infections, and saving lives. </p>

<p dir="ltr">Over the last century, cancer immunotherapy has become an attractive field due to the popularity of checkpoint blockades and adoptive cell therapy. Though these new frontier therapeutics are effective for certain populations, they’ve had either adverse effects on others or are non-efficacious when used to treat “cold tumors”. Hence, newer strategies are needed to sensitize cold tumors into immune-responsive “hot tumors”, which synergize with checkpoint blockades. The cyclic GMP-AMP synthase-Stimulator of INterferon Genes (cGAS-STING) pathway has been identified as a pathway that can initiate T cell infiltration and turn cold tumors into hot tumors. Therefore, STING agonists have been identified as potential remedies that could help bend the curve to increase the survival rate of cancer patients if combined with anti-PD1 and anti-CTLA4 therapies.</p><p dir="ltr">2’3’-cGAMP is a master regulator of the innate immune system and is produced by cGAS upon cancer deregulation as well as bacterial and viral infection. Although 2’3’-cGAMP is a nanomolar affinity binder to STING and has vast immunostimulatory potential, it is plagued by several limitations that prevent its use in vivo. Most medicinal chemists have focused on making phosphorothioate derivatives which circumvents 2’3’-cGAMP’s limitations, but synthesizing these analogs presents a synthetic challenge. Also, these derivatives are commonly administered via intratumoral injection, which is not an attractive mode of delivery. This dissertation tries to address some of these challenges and provide a newer platform to develop CDN-based STING agonists.</p><p dir="ltr">We describe a novel class of phosphorothioester-linked cyclic dinucleotides (endo-S-CDNs) as excellent STING agonists. Showing through structural-activity relationship (SAR) which groups are tolerated or detrimental for STING binding and cellular activity. Also, determining that these 5’-phosphorothioester-linked CDNs are resistant to cleavage by clinically relevant phosphodiesterases (PDEs). Finally, we discuss how this novel class of CDNs is suitable for subcutaneous dosing to clear tumors in different mouse models.</p>