Preventing Death: An old Drug Repurposed
One thing is certain for all living beings. At some point we will all die. This is not only true for whole organisms but also for the cells inside those organisms. Cells can die in multiple ways which can include age-related death, as seen for blood cells like neutrophils. Other cells die during development for the greater good; a prominent example is the death of cells between your fingers which shape these delicate parts of your body. Excitingly, murder is also part of the constant death in our bodies: for example, cytotoxic T cells patrol our bodies to watch for infected cells. If they find some, they will kill those by the so-called kiss-of-death. Although all of these are very different forms of death, one common thing is that the death happens in silence. A process termed apoptosis starts in which the cell is digested intrinsically. Proteins and DNA are cleaved and packaged into compartments which are called apoptotic bodies. Other cells, macrophages, recognize those and clean the mess up as soon as possible with no traces left behind. This process works to ensure that these forms of cellular death cause minimal collateral damage to other healthy cells.
However, there are also forms of cellular death which leave a mess. Pyroptosis is a prime example. This process happens when certain bacteria enter the cells, such as Shigella, Salmonella enterica and others. Their detection through inflammasomes such as NLRP3 leads to an activation of caspases that start cleaving certain proteins, such as inflammatory molecules like IL-1b and IL-18. This process also activates Gasdermin D, which is normally dormant in its full-length form. However, as soon as it gets cleaved the N-terminus can form pores in the cellular membrane. Through these pores, cytosolic proteins can exit the cell, including the freshly cleaved and activated IL-1b and IL-18. Additionally, these pores also allow water to enter the cell which leads to cell swelling and membrane rupture. These events induce a massive inflammation with recruitment and activation of immune cells which stands in stark contrast to the silent form of cell death as seen in apoptosis.
Pyroptosis has been associated with a variety of pathologies, among them cardiovascular diseases, neuronal diseases, diseases affecting the gastrointestinal tract and sepsis. Most recently, increased pyroptosis has been linked to the severe inflammatory syndrome caused by SARS-CoV-2. Thus, inhibition of pyroptosis and the associated secretion of pro-inflammatory molecules is a very relevant goal. So far, the pathway of pyroptosis was targeted either through biological drugs affecting IL-1b and/or IL-18, or through the inhibition of the inflammasome NLRP3. The caveats of these approaches are that, there are other inflammasomes that can trigger pyroptosis, and that pyroptosis leads to more than just secretion of these two cytokines IL-1b and IL-18. Thus, inhibition of Gasdermin D as the central protein in pore formation during pyroptosis seems to be a desirable upstream target.
In a recent Nature Immunology paper, a group of scientists led by Hao Wu tackled this goal. They designed a very elegant screen in which they incubated Gasdermin D with Caspase-11, which is the caspase that is responsible to cleave and activate Gasdermin D, and added liposomes which were loaded with terbium. Successful pore formation into these liposomes resulted in leakage of terbium which in turn bound to dipicolinic acid and with that generated fluorescence which could be detected with a simple plate reader. With this setup the scientists screened 3,752 small molecules for a potential inhibition of fluorescent signal and with it inhibition of Gasdermin D-mediated pore formation. 22 candidate compounds were identified which the authors of the paper then validated and tested for specificity in multiple experimental setups. The most prominent compound that was found to not only inhibit liposome leakage in vitro, but also specifically pyroptosis in cellular system was disulfiram. Disulfiram is a drug used for decades in the treatment of chronic alcohol abuse and thus has demonstrated safety in clinical use. The authors followed this exciting finding with a thorough analysis of how disulfiram is specifically inhibiting Gasdermin D. They found that it inhibits a reactive cysteine of Gasdermin D through attaching half of the symmetrical disulfiram molecule. In their final set of experiments the authors proofed the efficiency of the drug in vivo. Mice challenged with different doses of LPS, a model of sepsis, were either treated or not treated with disulfiram. Treated mice showed prolonged and increased survival in comparison to controls. This paralleled with a reduction in serum cytokines. Next, the scientists wanted to mimic clinical settings. Thus, LPS-challenged mice were only treated with disulfiram after a 12-hour delay. This also led to pro-longed survival, though all mice were dead after the experiment. Finally, they added copper gluconate which stabilizes disulfiram through complexation in the mix. When mice were challenged with LPS, treated with disulfiram and copper gluconate after a 12-hour delay, survival could not only be further prolonged, but a fraction of mice also survived the challenge.
In summary, the work of the Wu lab identifies a very promising drug inhibiting inflammatory cell death, disulfiram, that shows potential in the treatment of sepsis in mice. “This research discovery is coincidentally very timely today because most people think that the clinical deterioration of COVID-19 patients is mediated by a cytokine storm, or excessive release of inflammatory molecules,” says Judy Lieberman, one of the corresponding authors. As disulfiram has been used for decades in the clinic, safety has been well established. This drug then has great potential in the treatment of inflammatory disorders and syndromes.
By Nina Serwas
Further reading:
Hu, J. J. et al. FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation. Nature Immunology 21, (2020).
Schmidt, F. I. & Latz, E. Jack of all trades inhibits inflammation (in sober people). Nature Immunology 21, 716–726 (2020).
Yap, J. K. Y., Moriyama, M. & Iwasaki, A. Inflammasomes and Pyroptosis as Therapeutic Targets for COVID-19. The Journal of Immunology 089992, ji2000513 (2020).
Lawrence, S. M., Corriden, R. & Nizet, V. How Neutrophils Meet Their End. Trends in Immunology 1–14 (2020) doi:10.1016/j.it.2020.03.008.
Schema showing how disulfiram interrupts the inflammatory cascade. Illustration: Boston Children's