REGULATION OF THE CGAS-STING PATHWAY IN LUPUS August 10, 2017 REGULATION OF THE CGAS-STING PATHWAY IN LUPUS Monique Mendez Lairson Lab P.I.: Luke Lairson Mentor: Emily Chin Hello, my name is Monique Mendez and over these past several weeks, I had the chance to intern at the Lairson lab. Today I will be going over the Regulation of the cGAS-STING  Pathway and focus on what research I have conducted while here at Scripps.

Lairson Lab Chemical biology Drug discovery Screening of small molecule activators/inhibitors The Lairson lab specializes in chemical biology, drug discovery, and high-throughput screening in order to find small molecule activators and inhibitors. Currently, they are screening for small molecule activators to inhibit the cGAS-STING pathway.

Background: cGAS-STING Pathway Helps protect us from spread of viral infection Does this by increasing interferon production IFNs activate an inflammatory response So, for many reasons, the activation of the cGAS-STING pathway is still important as the pathway helps protect us from the spread of viral infection - it does so by stimulating the interferon pathway. For those of you who may not know, an interferon is a group of signaling proteins made and released by our body cells as a defensive response to viruses and bacteria, shielding us from infections And to give you guys a better understanding on how the pathway works: We first need dsDNA to bind to cGAS This bond then uses ATP and GTP as substrates to form cGAMP cGAMP binds to STING and this activates the transcription factor IRF3 IRF3 ultimately causes higher levels of IFN β (beta) and IFN α (alpha) expression However, as I said, our lab is interested in discovering a small molecule inhibitor because patients with lupus have cGAS-STING signaling and when their IFN pathway is activated, inflammatory responses are the outcome. Schematic of cGAS-STING pathway

Chen, Q. et al. Nat Immunol. (2016) What causes Lupus? Chen, Q. et al. Nat Immunol. (2016) Exonucleases prevent accumulation of DNA in the cytoplasm Mutations identified in patients with SLE Now, what causes Lupus? Well, in all of our bodies, we have exonucleases. An exonuclease is an enzyme that works by splitting nucleotides one at a time starting from the end of a polynucleotide chain. When it comes to those suffering from Lupus, TREX1 works exactly that way; by degrading and digesting all DNA that has been spilled out of the nucleus into the cytoplasm. DNA that has now been spread throughout the cytosol is now called cytosolic DNA. A common feature of Lupus, is the elevated expression of ifn-stimulated genes (ISGs), suggesting that a defect in clearing the cytosolic dna leads to the activation of the IFN pathway This starts the recurring cycle of the cGAS-STING pathway. Individuals with loss-of-function mutations in TREX1, only have the enzyme working partially, this is due to the sequence being folded improperly.

What is Lupus? Systemic Lupus Erythematosus (SLE): Symptoms: Affects approximately 1.5 million individuals in the USA Most commonly affects more females than males with a 9-to-1 ratio Symptoms: Inflammation of connective tissues such as cartilage and the lining of blood vessels affecting: Skin, joints, kidneys, lungs, central nervous system, and hematopoietic system Treatment: Current treatment options are focused on managing inflammation An inhibitor of the cGAS-STING pathway would reduce the source of inflammation Lupus is an auto-imflammatory disease. An autoimmune disease is a disease in which the body’s immune system attacks healthy cells. The technical term for Lupus is Systemic Lupus Erythematosus. Lupus affects approximately 322,000 to 1.5 million individuals in the U.S. It has been reported to affect more women than men with a ratio of 9-to-1. With patients of Lupus, the musculoskeletal, cardiovascular, hematopoietic and nervous system is affected. In essence, lupus causes inflammation and rashes throughout the body affecting a person’s joints, skin, kidneys, blood cells, brain, heart and lungs. Currently, treatment options are more focused towards managing inflammation. If we are successful in finding a small molecule inhibitor for the cGAS-STING pathway, we could reduce the source of inflammation.

Project Goals To determine the specificity of candidate inhibitors (identified through high-throughput screening) To determine if they inhibit cGAS directly, we will develop a cGAS enzymatic assay 2 cGAS + dsDNA + 2PPi Disappearance of substrate Appearance of product Throughout our research, our goal was to determine whether or not specific candidate inhibitors directly target the cGAS protein or STING protein. In order to do so, we looked at the disappearance of ATP concentration and appearance of cGAMP concentration.

Experimental Methods Quantification of 2’3’-cGAMP by HPLC Luminescence Assay During our project, we involved numerous experimental methods: *One of the tests we conducted was through measurement of production of cGAMP to see if cGAS would bind to STING. To quantify the production of cGAMP, we used an HPLC machine. We also performed a luminescence assay with Quanti-Luc in order to measure ATP concentration.

Quantification of 2’3’-cGAMP through HPLC We identified optimized enzymatic cGAS reaction conditions For the first part of our results, we were able to identify optimized enzymatic cGAS reactions. We were able to optimize reaction conditions in order to measure the changes in ATP concentration We also optimized reaction conditions to detect the appearance of 2’3’ cGAMP through use of HPLC.

Quantification of ATP using Kinase Glo Max reagent We optimized the assay to measure changes in ATP concentration as an output for cGAS enzymatic activity. R² = 0.9948 We optimized the assay to measure changes in ATP concentration as an output for cGAS enzymatic activity. In our first chart, our data shows that we were able to optimize cGAS enzymatic activity through an ATP standard curve with Kinase Glo Max. We measured the production of cGAMP in order to see if it would bind to STING. Using a dilution series of ATP we were able to generate a linear standard curve as demonstrated on the left panel with an r2 value of .99. (r2 value is a statistical measurement of how close the data set fits to regression line) In the second chart, is our controls and how much their ATP levels varied

Conclusions/Future Directions We can measure cGAS enzymatic activity through: quantification of ATP concentration detection of cGAMP by HPLC This assay is optimized for testing putative inhibitors of cGAS Going forward, we can now measure cGAS enzymatic activity through: Quantification of ATP concentration and through detection of cGAMP by use of an HPLC machine. Our assay is now optimized for testing accepted inhibitors of cGAS

Acknowledgements I would like to thank: Emily Chin Luke Lairson Members of the Lairson lab Ashley Hull Dona Mapston Alexander Becker Before I conclude my presentation, I would like to thank my mentor Emily Chin, my P.I. Luke Lairson, and the rest of the members of the Lairson lab. I would also like to thank our program coordinators, Ashely Hull of the Scripps Graduate Office, Dona Mapston from the Salk Institute and Alexander Becker from the San Diego workforce.