04 Jan Second Protein Target of Aspirin Metabolite Identified
MedicalResearch.com Interview with:
Prof. Daniel F. Klessig
Boyce Thompson Institute for Plant Research,
Department of Plant Pathology and Plant-Microbe Biology
Cornell University, Ithaca, New York
MedicalResearch: What is the background for this study?
Prof. Klessig: Acetyl salicylic acid, commonly called aspirin, has been the most widely used drug worldwide for more than a century. Currently, 80 million pounds of aspirin are produced worldwide every year and almost 30 billion tablets are consumed annually in the US alone. Long before German pharmacologist Johann Buchner identified the salicylic acid derivative salicin in 1828 as the ingredient in willow bark that is responsible for its therapeutic effects, different cultures throughout the world were, and many still are, using a variety of plants rich in salicylic acid derivatives, such as willow, wintergreen, and meadowsweet, to treat pain, fever, swelling, and other maladies. Aspirin also is used to reduce the risk of heart attack, stroke, and certain cancers.
One might expect that aspirin’s mechanisms of action would be well understood, given its extraordinarily widespread use and the fact that it was first synthesized by the Bayer chemist Felix Hoffmann over 100 years ago. The prevailing view in the biomedical community has been that aspirin works primarily, if not exclusively, by irreversibly inhibiting the enzymatic activities of cyclooxygenases 1 and 2 (COX1 and COX2), thereby disrupting the synthesis of inflammation-inducing prostaglandins. However, this assumption ignores two important facts.
- First, aspirin is rapidly converted to salicylic acid (SA) in the body. Indeed, almost all aspirin is metabolized to SA within an hour after ingestion.
- Second, SA and many of its natural plant derivatives are rather poor inhibitors of COX1 and COX2 as compared to aspirin, yet SA and aspirin have nearly the same beneficial pharmacological effects. Thus, there must be additional targets through which aspirin/SA exerts its many effects. Over the past two decades, a number of proteins whose activities are altered by aspirin/SA have been identified; however, their relevance as aspirin/SA targets has been called into question due to the very high, non-physiological levels of aspirin/SA required to alter their activities.
In light of our unexpected discovery that SA mediates its physiological effects in plants via many targets, and given that SA is a key hormone produced by all plants, we hypothesized that there might be multiple targets through which SA acts in animals, regardless of whether it is obtained in low to moderate levels via the diet or in moderate to high doses through herbal-based medicines or aspirin usage.
MedicalResearch: What are the main findings?
Prof. Klessig: To investigate whether aspirin/SA exerts its pharmacological activities by targeting proteins besides the cyclooxygenases in humans, we used high-throughput approaches developed to identify proteins that mediate SA signaling in plant immunity. This strategy identified several proteins that bind SA and as a result they exhibit altered activity, including Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH). In the body, GAPDH is a cytosolic enzyme that plays a central role in the production of energy, via a metabolic process called glycolysis. In addition to this housekeeping role, other functions of this protein have come to light in the past few decades, including its participation in DNA repair and transcription. It has RNA and DNA binding activities, which interestingly have been usurped by viruses for their replication, including human hepatitis A, B, and C viruses and tomato bushy stunt virus. In addition, numerous studies have linked GAPDH with cell death/apoptosis induced by a variety of agents, including N-methyl-N-nitroso-N1-nitroguanidine (MNNG, a DNA alkylating agent which mimics neuronal cell death induced by nitric oxide). Thus, GAPDH is a major suspect in neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and Huntington’s diseases. The pioneering work of Ishitani and Chuang and later Snyder and co-workers revealed the central role GAPDH plays in neurodegeneration. Moreover, in 2006 Snyder and colleagues discovered that GAPDH, along with the nitric oxide and the E3 ubiquitin ligase Siah, are involved in a novel cell death cascade. In brief, oxidative stress conditions can lead to elevated levels of nitric oxide, which cause S-nitrosylation of GAPDH’s catalytic cysteine 152, which inactivates its glycolytic activity and induces its interaction with Siah. GAPDH bound to Siah is then translocation to the nucleus, where it stabilizes this E3 ubiquitin ligase. This results in increased turnover of Siah’s nuclear target proteins leading to cell death. The anti-Parkinson’s disease drug deprenyl, which reduces neuronal cell death in both in vitro and in vivo models, prevents S-nitrosylation of GAPDH, blocks the GAPDH-Siah interaction, and inhibits GAPDH nuclear translocation.
We discovered that human GAPDH, like its plant counterpart, binds SA. In collaboration with Solomon Snyder’s group SA was found to suppresses both GAPDH nuclear translocation and cell death induced by MNNG. Importantly, we also identified several natural and synthetic derivatives of SA that bind to GAPDH more tightly than aspirin/SA and inhibit its translocation to the nucleus and the resulting cell death more effectively than SA. Interestingly, these natural derivatives are produced by an herb used in traditional Chinese medicine Glycyrrhiza foetida (licorice), while the synthetic derivative was designed based on both the structure of the herbal derivative and our studies of the binding activities of other SA-like compounds with GAPDH and the other new target of SA/aspirin we recently discovered – High Mobility Group Box 1 (HMGB1; see Choi et al., 2015, Molecular Medicine 21:526-535).
MedicalResearch: What should clinicians and patients take away from your report?
Prof. Klessig: The discovery that GAPDH is another target of SA/aspirin improves our understanding of how aspirin works, but it should not change the way doctors presently prescribe aspirin or the way patients take it. While the results from our study are exciting and provide great promise as a possible treatment for neurodegenerative diseases, they will need to be followed by much more comprehensive studies, including mouse model work and human clinical studies. However, our ability to design new molecules that bind to and inhibit the bioactivities of both GAPDH and HMGB1 much more effectively than SA or aspirin provides proof of concept that it is possible to design a “better aspirin”. Development of these drugs will require the interest and the investment of pharmaceutical companies, because this work is very expensive and goes far beyond the budget of an academic lab. Although developing these drugs will take several years, the creation of new classes of drugs to combat neurodegeneration (GAPDH) and/or inflammation and its associated diseases such as cancer and arthritis (HMGB1) will certainly be very big news.
MedicalResearch: What recommendations do you have for future research as a result of this study?
Prof. Klessig: GAPDH and HMGB1 are only the first two of several novel targets of aspirin/SA in humans that we have uncovered. Another target is a key regulator of energy metabolism, and therefore is an important player in obesity and diabetes. Several additional likely targets require more study, including a protein that, like HMGB1, plays a role in regulating inflammation. Further research will likely reveal additional targets of aspirin/SA and clarify which are responsible for SA’s/aspirin’s therapeutic activity, as well as their negative side effects. Given the potentially large number of targets for aspirin/SA, the multiple pharmacological effects of these compounds, and the widespread use of aspirin and/or natural SA-based derivatives, which our studies suggest are the basis for at least some traditional medicines, it behooves the biomedical community, including the US National Institutes of Health and pharmaceutical companies, to invest in studies that clarify exactly how aspirin/SA works – that is, elucidating their mechanisms of action. I predict that aspirin/SA will exert their effects in humans via multiple mechanisms of action. In light of our discovery of more than two dozen proteins in plants, through which SA mediates its multiple effects on immunity and several other plant processes, and that most animals eat plants, all of which contain SA as an important hormone, a large numbers of targets in human perhaps should not be surprising. Interestingly, many of these SA targets are shared by plants and humans/animals, including the GAPDHs and HMGBs. For example, both human HMGB1 and its plant counterpart HMGB3, when released from damaged cells into the extracellular space, induce innate immune responses to protect the damaged tissue against infection. SA binds to both and inhibits their immune/inflammatory activities.
Prof. Daniel F. Klessig (2016). Second Enzyme Target of Aspirin Metabolite Identified