The cells of our body are exposed to many different threats, including viral infections, cancer and accidents in their power plants (mitochondria). What all these threats have in common is that they cause DNA double strands (dsDNA) to appear in the cell's plasma – where they do not belong and where their presence as foreign genetic information signals maximum danger. Even our own dsDNA should not be present outside the cell nucleus and mitochondria. How our innate immune system recognizes and fends off the danger of DNA in the wrong place remained a mystery for a long time. The three prizewinners solved it between 2008 and 2013 and have since clarified it more and more comprehensively. They discovered a signaling pathway that starts with an enzymatic sensor, which clasps on to dsDNA as soon as it detects it in the cytoplasm. The enzymatic sensor changes its shape in the process, enabling it to catalyze the formation of a molecular messenger. This messenger, in turn, triggers an intracellular receptor that receives and translates the messenger's dispatch by sending its own communication to certain genes in the cell nucleus, asking them to produce interferons immediately. These interferons spread to the surrounding tissue and call for help. The distinguishing feature of this so-called cGAS-STING pathway is its universality: Its sensor does not differentiate between external and endogenous dsDNA. This violates the rule that our immune system must clearly distinguish between "foreign" and "self" – a violation that is all the more risky as it harbors the possibility of unintentional self-destruction. It does, however, offer medicine a double opportunity to intervene therapeutically in this signaling pathway.

We are attacked by thousands of bacteria and viruses every day. In most cases, our body successfully fends off these attacks. This is thanks to its innate immune system, which keeps the intruders at bay until its signals have activated the body's acquired immune system, whose antibodies and T-cells eliminate the attackers – which can take several days. Without our innate immunity, we would hardly survive these days. Despite this, research into this immunity has long led a shadowy existence. While the basic features of acquired immunity were elucidated in great detail in the 20th century, it remained unclear for a long time how the innate immune system perceives microbial attacks. This only changed in the mid-1990s, with the discoveries made independently by Jules Hoffmann in flies, which do not have an acquired immune system, and Bruce Beutler in mice. These findings showed that cells involved in innate immune defense have receptors on their surface (pattern recognition receptors = PRRs) with which they recognize molecular patterns that are typical of pathogens such as bacteria and viruses, but do not normally occur in the organism under attack (pathogen-associated molecular patterns = PAMPs). The binding of a PAMP to a PRR triggers a signaling cascade within the affected cell, which in turn activates various components of innate immunity. The first PRRs discovered by the later Nobel Prize laureates Hoffmann and Beutler were toll-like receptors (TLRs). Incidentally, they were so named because they resemble the protein whose effect on the embryonic development of the Drosophila fruit fly Nobel Prize winner Christiane Nüßlein-Vollhard (Physiology or Medicine, 1995) once described with the words "Das ist toll!" [“That is amazing!"].

One hundred years of perplexity
The discovery of the first PRRs gave a huge boost to research into innate immunity, and over the course of several years led to the identification of many more TLRs and other PRR families, including receptors that are not anchored in the cell membrane but patrol its plasma to ward off pathogens that have already invaded the cell interior. These include NOD-like receptors, specialized on bacteria, and RIG-I-like receptors, specialized on the RNA of viruses. All insights notwithstanding however, the question of how DNA is recognized in the cytoplasm and triggers an immune response remained unanswered – even though Ilya Mechnikov had already reported of its ability to do so in 1908, when he was awarded the Nobel Prize for Physiology or Medicine, which he shared with Paul Ehrlich. It was not until one hundred years later that the fog surrounding the answer to this question began to clear.

The discovery of STING
In 2008, virologist Glen Barber and his team at the University of Miami shed light onto this conundrum. Based on the knowledge that PRR signaling cascades lead to the release of interferon-beta (IFN-ß), he set out in search of previously unknown intracellular initiators of IFN-ß production. To this end, he applied expression cloning and introduced a total of 14,500 different proteins individually into a cell culture's cells, coupling their IFN-ß genes with enzymes that cause cells in which IFN-ß is produced to light up. This enabled him to measure the strength of the immune response to each protein. The top places in the hit list of his series of experiments went to some molecules from familiar PRR cascades. However, a previously unknown protein stood out among the runners-up. Barber isolated it and showed that, with 379 amino acids, it winds its way through the membrane of the endoplasmic reticulum (ER) several times. He investigated its function in other cell cultures and knock-out models and was able to confirm that it is essential for the production of IFN-ß and other cytokines after infection with DNA viruses such as herpes simplex. It is for this reason that Barber aptly named it STING: stimulator of interferon genes.

In search of the sensor
Although the available experimental evidence clearly demonstrated STING's essential role in the immune response to dsDNA in the cytoplasm, it was obvious that STING is not directly activated by dsDNA. It is, after all, a signal transducer, not a sensor. It was Zhijian 'James' Chen of the University of Texas Southwestern Medical Center in Dallas and his team who discovered this sensor and its messenger, which activates STING, by ingeniously combining highly sensitive biochemical methods with tremendous intuition. Chen first introduced different types of DNA into the cells of a culture. Using RNA interference, he blocked all signals emanating from STING, and then drained the treated plasma from the cells of this culture to obtain a cell-free extract. He mixed this extract with cells from another culture, whose membrane he chemically perforated so that the plasma could flow freely in and out, but whose organelles remained positioned intact intracellularly. If a molecule is formed in the DNA-treated STING-blocked extract that transmits a message to STING, Chen reasoned, it will then activate the unblocked STING in the perforated cells and cause the formation of interferons there. The hypothesis turned out to be true. Surprisingly, this messenger molecule remained active even after it was heated to 95 degrees Celsius, a temperature almost no protein can survive. The experiment showed: The messenger is the small ring-shaped dinucleotide cGAMP – hence the enzyme that enables the formation of cGAMP had to be the DNA sensor they were looking for. In an extract that is full of other proteins, however, this cGAMP synthase (cGAS) is only present in such vanishingly small quantities that it was an extraordinary biochemical feat to purify, isolate and characterize it as a protein comprising 522 amino acids.

A very special messenger
In December 2012, the journal Science published Chen's discovery of the DNA sensor cGAS and the dinucleotide cGAMP as its second messenger ahead of print. About four months later, detailed descriptions of the production process and chemical structure of the messenger substance cGAMP catalyzed by cGAS were published. It differed from previously known bacterial dinucleotides, both in the way it was produced and in the binding between its two building blocks. This unique characteristic was decoded by Andrea Ablasser just a few years after having completed her medical studies. She was awarded the Paul Ehrlich and Ludwig Darmstädter Early Career Award 2014 for this achievement.

A milestone in immunological research
The discovery of the cGAS-STING signaling pathway marked a milestone in the study of innate immunity. Starting from this milestone, all three prizewinners proceeded to map the branches of the pathway they discovered in ever greater detail, thus opening up previously hidden therapeutic paths to medicine. When cGAS detects dsDNA in the cell plasma, two of its molecules bind to each of two DNA strands. This creates ladder-like structures that separate into liquid droplets, which are necessary for cGAS to open previously inaccessible reaction pockets. GTP and ATP come together in these pockets to form the messenger molecule cGAMP in a two-stage process. cGAMP then flows around STING, and also reaches neighboring cells via channels between cells – so-called gap junctions. The binding of cGAMP deforms the STING receptors in such a way that they detach from the ER membrane and swim to the intracellular chemical factories closest to them, the Golgi apparatus. There, a fatty acid is attached to them, which enables them to include waiting signal molecules in the alarm relay by labeling them with phosphate groups. The target of this relay is the chromosomes in the cell nucleus on which the genes are located, according to whose plan IFN-ß and other cytokines are produced. These molecules then mediate the immune system's response to microbial attacks by stimulating the activity of macrophages, dendritic cells and natural killer cells, among others. They are also active against tumors.

Defensive readiness with risk
The cGAS-STING signaling pathway is ancient. Over millions of years, it has remained virtually unchanged in most multicellular organisms. Because cGAS interacts with every dsDNA regardless of its sequence, it always jumps into the breach very quickly and reliably. Accordingly, it also shows this immediate defensive readiness against the body's own dsDNA. To minimize the resulting risk of being attacked by our own immune system, evolution has built into our cells regulatory mechanisms for self-protection. Under normal conditions, our own DNA is strictly compartmentalized: it only occurs in the cell nucleus and in the mitochondria. When the boundary between the nucleus and plasma temporarily dissolves during cell division (mitosis), our own DNA remains shielded by its chromatin packaging and is thus protected from access by cGAS. Furthermore, specialized enzymes (nucleases) ensure the continuous degradation of misplaced DNA. There are also threshold values for their concentration and strand length that dsDNA must reach in the cytoplasm in order to trigger cGAS. These values are reached less frequently by the cell's own dsDNA than by foreign dsDNA. However, nature obviously did not plan for us humans to live as long as we do today. The older we get, the more frequently the regulatory mechanisms to protect our own DNA from cGAS fail. “The genes of the cGAS-STING signaling pathway are pleiotropic genes that are important for survival in the beginning, but can lead to diseases in later years as well as due to changes in our lifestyle," explains Andrea Ablasser.

Two therapeutic options
Hereditary forms of Parkinson's disease, for example, are associated with an inability of certain nerve cells to dispose of damaged mitochondria. As a result, mitochondrial DNA accumulates in the cytoplasm. This alerts the cGAS sensor, which then triggers a destructive inflammation in the nerve tissue via cGAMP and STING as an immune response. In older people in particular, activation of the cGAS-STING pathway probably underlies many so-called sterile inflammations that are not caused by an infection. In addition to neurodegenerative diseases, such inflammations can presumably also cause heart failure, diabetes and other autoimmune diseases. The development of substances that inhibit the cGAS-STING signaling pathway therefore has great therapeutic potential. Andrea Ablasser and her group succeeded in synthesizing the first STING inhibitor in 2018.

Conversely, agonists of the cGAS-STING pathway are already being tested preclinically as cancer drugs. They show strong anti-tumor effects in animal experiments, especially in combination with checkpoint inhibitors. This is because, due to their high division rate, the genome of cancer cells is usually not very stable, leading to the appearance of endogenous dsDNA in the cytoplasm. As a result, the cGAS-STING pathway kicks in and enables the body to eliminate cancer cells by producing interferons. In addition, cGAS-STING can suppress tumor development by putting cancer cells into a state of senescence, i.e. permanently inhibiting their ability to divide. However, caution is required when using agonists pharmacologically, as some tumors have learned to use the cGAS-STING pathway to their advantage. The risk of a cytokine storm must also be kept as low as possible.

Even if the path to approved antagonists and/or agonists of cGAS or STING is still a great one, it has been taken by many pharmaceutical companies. With its structurally clearly defined target molecules, the signaling pathway discovered and decoded by the prizewinners is one of the most attractive challenges in current pharmaceutical research.