Evaluation of cyclic dinucleotides and their prodrugs in biochemical and cellular assays

Özdemir, Dilara (2023): Evaluation of cyclic dinucleotides and their prodrugs in biochemical and cellular assays. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

The innate immune pathways gained more importance since the discovery of the extended role of type I interferons. Type I interferons are produced primarily upon viral and bacterial invasions and function by inducing immune responses. As a result, innate and adaptive immune cells are differentiated, activated and recruited to the pathogen-invaded cells for destruction. Type I interferons are also produced by cancer cells and immune cells regulating the cancer immunity cycle. The cGAS-STING pathway is one of the key pathways resulting in type I interferon mediated immune response. The cGAS-STING pathway is activated by double-stranded DNA (dsDNA) leaking into the cytosol. When cGAS (cytosolic-Guanosine-Adenosine-Synthase) binds to dsDNA, ATP and a GTP are utilised for the production of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). cGAMP binds to STING (Stimulator of Interferon Genes), initiating a signalling cascade. The high rate of chromosomal instabilities in cancer cells as well as the instability of genetic material in aged cells are reasons for dsDNA to leak out from the nucleus and localise in the cytosol. Hence, the cGAS-STING pathway is activated and immune cells are recruited to the site of danger. In recent years, targeting the cGAS STING pathway has been an emerging strategy for drug discovery and led to the development of cGAMP analogues. The group of Carell has designed and synthesised cyclic dinucleotides as STING agonists. In the first part of this thesis, the cyclic dinucleotides and their developed prodrugs were evaluated using cellular and biochemical assays. Their rate of inducing type I interferon production was monitored in THP 1 monocytic cells. Their effective concentration 50 (EC50) were measured and promising cGAMP analogues were tested for their stability against currently known cGAMP degrading enzymes. Two compounds, the 2’,3’-dideoxy-cGAMP and 2’,3’-dideoxy-cAAMP, were identified to be stable against poxins, which are viral enzymes from the poxvirus family. Two prodrugs of 2’,3’-dideoxy-cAAMP were synthetically developed by the group of Carell, one of them containing the SATE linkers, named PRO1, another one bearing an additional photocleavable group named PC-PRO1. These were also evaluated regarding their rate of type I interferon production. With an EC50 value of 49 nM, PRO1 shows a 1500-fold increase in interferon production in THP-1 monocytic cells compared to its precursor 2’,3’ dideoxy cAAMP and a 200-fold increase compared to natural cGAMP (2’,3’ cGAMP). Overactivation of STING and overproduction of type I interferons is related to specific diseases, aging, and metastatic activity in cancers. Therefore, besides STING agonists, STING antagonists also carry a therapeutic potential. For the development of a STING antagonist, G. Ganazzoli from the Carell group used the PROTAC (Proteolysis Targeting Chimera) approach utilising 2’,3’-dideoxy-cGAMP as a STING recruiter. This compound named PROTAC1, was evaluated in the second part of this thesis for its ability to degrade STING in different time frames. The analysis was done on THP-1 monocytic cells and western blotting was used in which the degree of STING degradation was measured by the intensity of the STING-antibody bound bands. As an internal control, the CoxIV of each sample was targeted and their band intensities were used for normalisation of the STING signal. A successful degradation of STING in THP-1 monocytic cells was observed when 25-50 nM of PROTAC1 was applied to the cells for 16 hours. In the published work in section 5 of this thesis, the chemical synthesis of a fluorescent cGAMP analogue, cthGAMP, was introduced. The successful entry of cthGAMP was observed in THP-1 monocytic cells with two-photon excitation microscopy. Interferon production was observed when cthGAMP was transfected in the THP-1 monocytic cells, providing proof of biological activity. This compound was suggested for assay development purposes concerning the cGAS-STING pathway, due to its scalability of its synthetic route and biological activity. In the second research article presented in section 5, the supply mechanism of α-ketoglutarate to the dioxygenase TET3 by the metabolic enzyme Gdh was described in hippocampal neurons for the oxidation process of 5’ methylcytosine. The oxidation of 5’ methylcstosine to 5’ hydroxymethylcytosine was observed upon Gdh localisation in the nucleus together with TET3. A functional Gdh was required for this conversion in activated hippocampal neurons. In this study, HEK293 cells were used for expression and co-expression of natural and modified enzymes, which were used for immunocytochemistry experiments. Hippocampus slices from mice were used for neural activation, immunohistochemistry and proximity ligation assays. Observations were made by confocal microscopy, whereas quantification of 5’ methylcytosine and its oxidation products were done by UHPLC-QQQ-MS.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: cGAMP, STING, Cyclic Dinucleotides
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 17. Februar 2023
• 1. Bericht­erstatter:in: Carell, Thomas