Abstract The plant hormone salicylic acid (SA) controls several physiological processes and is a key regulator of multiple levels of plant immunity. To decipher the mechanisms through which SA’s multiple physiological effects are mediated, particularly in immunity, two high-throughput screens were developed to identify SA-binding proteins (SABPs). Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) from plants (Arabidopsis thaliana) was identified in these screens. Similar screens and subsequent analyses using SA analogs, in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology, established that human GAPDH (HsGAPDH) also binds SA. In addition to its central role in glycolysis, HsGAPDH participates in several pathological processes, including viral replication and neuronal cell death. The anti-Parkinson’s drug deprenyl has been shown to suppress nuclear translocation of HsGAPDH, an early step in cell death and the resulting cell death induced by the DNA alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine. Here, we demonstrate that SA, which is the primary metabolite of aspirin (acetyl SA) and is likely responsible for many of its pharmacological effects, also suppresses nuclear translocation of HsGAPDH and cell death. Analysis of two synthetic SA derivatives and two classes of compounds from the Chinese medicinal herb Glycyrrhiza foetida (licorice), glycyrrhizin and the SA-derivatives amorfrutins, revealed that they not only appear to bind HsGAPDH more tightly than SA, but also exhibit a greater ability to suppress translocation of HsGAPDH to the nucleus and cell death. 
 
 Citation: Choi HW, Tian M, Manohar M, Harraz MM, Park S-W, Schroeder FC, et al. (2015) Human GAPDH Is a Target of Aspirin’s Primary Metabolite Salicylic Acid and Its Derivatives. PLoS ONE 10(11): e0143447. https://doi.org/10.1371/journal.pone.0143447 Editor: Hua Lu, UMBC, UNITED STATES Received: August 14, 2015; Accepted: November 4, 2015; Published: November 25, 2015 Copyright: © 2015 Choi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information file. Funding: This work was supported by grants from the Arabidopsis 2010 Initiative of the US National Science Foundation (IOS-0820405 to D.F.K.) and US Public Health Service (MH18501 to S.H.S. and M.M.H.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. 
 
 Introduction Through most of human history, medicine has been based on plant remedies. Plant/herb-based medicine, sometimes referred to as traditional medicine (TM), still is the primary form of treatment for billions of people worldwide, particularly in developing countries, and is regaining favor in Western societies. Even in "modern" medicine, approximately half of the pharmaceuticals developed over the past 20 years and approved by the US FDA are natural products (mainly plant derived), or are synthetic derivatives of, or have at their core a prototype molecule derived from, natural products [1]. Salicylic acid (SA) and its derivatives, collectively termed salicylates, are a prime example. Acetyl SA, commonly called aspirin, has been the most widely used drug worldwide for the past two centuries—today Americans alone consume 80 million aspirin tablets daily. SA and its derivatives have been used for millennia to reduce pain, fever, and inflammation. Aspirin is also used to reduce the risk of heart attack, stroke, and certain cancers. The primary action of aspirin in mammals has been attributed to the disruption of eicosanoid biosynthesis through the irreversible inhibition via acetylation of cyclooxygenases (COX) 1 and 2, thereby altering the levels of prostaglandins and leukotrienes [2]. However, since aspirin is rapidly de-acetylated by esterases in the bloodstream (t 1/2 = 20 min), much of aspirin’s bioactivity can be attributed to its primary metabolite, SA, which reaches ~140 μM (~20 mg/L) within 1 h after intake of 500 mg of aspirin [3] and has a half-life of many hours in plasma [4]. Notably, although in vitro SA is a much weaker inhibitor of COX activity (IC 50 >100 mg/L ~500 μM) than aspirin (IC 50 = 6.3 mg/L ~35 μM), their anti-inflammatory effects are comparable in vivo [5,6]. Thus, there must be additional targets through which aspirin/SA brings about its many pharmacological effects. In plants, SA is involved in many physiological processes including immunity, where it plays a central role [7,8]. Over the past quarter century, we have sought to elucidate SA’s mechanism(s) of action by identifying an array of plant proteins that bind SA, and, as a result, exhibit altered activity, which in turn modulates immune responses. Recently, we developed high-throughput screens to identify additional SA-binding proteins (SABPs) that may be targets of SA. This has led to the discovery of dozens of novel SA targets in plants [9–11]. To investigate whether aspirin has additional targets in humans, besides the cyclooxygenases, that may be responsible for some of aspirin’s multiple pharmacological activities, we employed similar screens of cultured human cells and identified many new potential targets of SA. In addition, several human proteins were identified as SA targets based on the SA-binding activity of their plant counterparts (orthologs). Among these is Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH). GAPDH is a cytosolic homotetrameric 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 [12] and transcription [13]. It has RNA and DNA binding activities, which interestingly have been usurped by viruses for their replication, including hepatitis A, B, and C [14–17] and tomato bushy stunt virus [18]. In addition, numerous studies have linked GAPDH with cell death/apoptosis induced by a variety of methods such as treatment with N-methyl-(R)-salsolinol (a neurotoxin), staurosporine, MG132, reactive oxygen species (ROS) or N-methyl-N-nitroso-N1-nitroguanidine (MNNG, a DNA alkylating agent which mimics neuronal cell death induced by ROS; [19]) in multiple cell types including both neuronal and non-neuronal cells such as fibroblast and human embryonic kidney (HEK) 293 cells (reviewed in [20]). Thus, GAPDH is a major suspect in neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and Huntington’s diseases [20]. The pioneering work of Ishitani and Chuang [21] and later Snyder and co-workers [19] revealed the central role GAPDH plays in neurodegeneration. Moreover, in 2006 Snyder and colleagues discovered that GAPDH, along with the ROS nitric oxide (NO) and the E3 ubiquitin ligase Siah, are involved in a novel cell death cascade. In brief, oxidative stress conditions lead to elevated levels of NO, which cause S-nitrosylation of GAPDH’s catalytic cysteine 152, which inactivates its glycolytic activity and induces its interaction with Siah. The monomeric GAPDH bound to Siah then is 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-PD drug R-(-) deprenyl and related agent TCH346, which reduce neuronal cell death in both in vitro and in vivo models, prevent S-nitrosylation of GAPDH, block the GAPDH-Siah interaction, and inhibit GAPDH nuclear translocation [22]. Here we demonstrate that human GAPDH, like its plant counterpart, binds SA, as well as natural and synthetic derivatives of SA. Their binding suppresses both GAPDH nuclear translocation and cell death induced by MNNG. 
 
 Materials and Methods Chemicals and anti-SA antibody Salicylic acid (SA)(Cat#: 247588), Aspirin (Cat#: A5376) and Glycyrrhizin (Cat#: 50531) were purchased from Sigma-Aldrich. The synthesis of amorfrutin B1, FN1, and FN2 was described previously [23]. N-Methyl-N-nitroso-N'-nitroguanidine (MNNG) (Cat#: N-12560) was purchased from Chem Service Inc. Anti-SA antibodies were obtained from Acris Antibodies (Cat #: AP09810PU-L). Plasmid construction, expression and purification of His-tagged HsGAPDH The plasmid pET28a-HsGAPDH used to express N-terminally His-tagged human (Homo sapiens) GAPDH (HsGAPDH) in E. coli was constructed by cloning the PCR-amplified protein-encoding sequence of human GAPDH (GenBank: M17851.1) into EcoRI and SalI sites of pET28a. The primers used are F: 5’- gcggaattcATGGGGAAGGTGAAGGTCGGAG -3’ and R: 5’- gcggtcgacTTACTCCTTGGAGGCCATGTGG -3’. Gene-specific sequences are in upper case and the restriction sites are underlined. cDNAs prepared from Hela cells were used for PCR amplification and cloning. The insert of the resultant plasmid was sequenced to confirm the accuracy. There was 1 nucleotide difference from the sequence in GenBank (M17851.1) without leading to any amino acid change. His-tagged recombinant HsGAPDH was expressed and purified in E. coli BL21(DE3) as described previously [24]. SA-binding activity analysis of HsGAPDH SA-binding activity analyses by photoaffinity labeling and surface plasmon resonance (SPR) were performed as described previously [9]. Synthesis of N-acetyl 3-(2-aminoethyl) salicylic acid Triethylamine (123 mg, 165 μL, 0.95 mmol) was added to a stirred solution of 3-(2-aminoethyl)salicylic acid (78 mg, 0.43 mmol) in a mixture of dimethylformamide (1.2 mL) and water (0.2 mL) at 0°C. After 5 min of stirring, a solution of acetic anhydride (46 mg, 42.5 μL, 0.45 mmol) in dimethylformamide (0.7 mL) was added dropwise with stirring over a time period of 5 min. After stirring at 20°C for 1 h, methanol (5 mL) was added and the mixture was evaporated to dryness in vacuo. The residue was chromatographed over silica using a Combiflash RF medium pressure automated chromatography system using a gradient of 5–40% methanol in dichloromethane as solvent. N-acetyl 3-(2-aminoethyl)salicylic acid was obtained as a colorless oil (purity >97% by 1H NMR spectroscopy) that solidified upon standing. Yield 45 mg (0.20 mmol). 1H NMR spectroscopy (methanol-d 4 , 600 MHz): δH 7.75 (dd, J = 8.0 Hz, 1.7 Hz, 1 H), 7.34 (dd, J = 7.4 Hz, 1.7 Hz, 1 H), 6.82 (dd, J = 8.0 Hz, 7.4 Hz, 1 H), 3.42 (t, J = 7.1 Hz, 2 H), 2.84 (dd, J = 7.1 Hz, 2 H), 1.99 (s, 3H). NMR spectroscopy was performed using a Varian INOVA 600 MHz NMR spectrometer (600 MHz 1H reference frequency) equipped with an HCN indirect-detection probe. High-resolution negative-ion electrospray mass spectrometry: observed m/z 222.0781 [M + H]-, calculated for C 11 H 12 NO 4 -, 222.0772. High-resolution UHPLC- (ultra high pressure liquid chromatography-) mass spectrometry was performed on a Thermo Scientific-Dionex Ultimate3000 UHPLC system equipped with a diode array detector and connected to a Thermo Scientific Q Exactive Orbitrap mass spectrometer operated in electrospray negative (ESI-) ionization mode. Evaluation of the potential binding of SA derivatives by HsGAPDH The potential binding between HsGAPDH and SA derivatives were evaluated by testing whether these derivatives were able to suppress the binding of HsGAPDH to 3AESA immobilized on CM5 sensor chip using SPR. The indicated concentrations of purified His-tagged HsGAPDH recombinant protein was mixed with 1 mM of various chemicals or the same amount of solvent, which were used to make chemical stock solution, in SPR running buffer [9], and then flown over 3AESA-immobilized CM5 sensor chip. The following data analysis was performed as determination of SA-binding activity using SPR [9]. Nuclear fraction isolation HEK 293 cells were pretreated with indicated concentrations of SA or other substances for 30 min then treated with 500 μM of N-Methyl-N-nitroso-N'-nitroguanidine (MNNG) for 30 min. Then the nuclear fraction was isolated as follows. The cells were scraped in ice cold hypotonic buffer containing 20 mM Tris-HCl, pH 7.4, 10 mM NaCl and 3 mM MgCl 2 . After a low speed (1,000 x g) spin for 5 min at 4°C, cells were resuspended in hypotonic buffer and incubated on ice for 15 min. NP-40 was then added to a final concentration of 0.5%. Nuclear fraction was pelleted by using a low speed (800 x g) spin for 10 min at 4°C. Nuclear fractionation using hypotonic buffer, NP-40 and centrifugation was repeated two more times as described above. Pellet was washed three times with ice cold phosphate buffer saline buffer and then resuspended in lysis buffer containing: 10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium fluoride (NaF), 20 mM Na 4 P 2 O 7 , 2 mM Na 3 VO 4 , 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, plus protease inhibitor cocktail containing; 0.5 μg/ml antipain, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml chymostatin and 1 μg/ml pepstatin A. The nuclear slate was cleared by centrifugation for 30 min at 10,000 x g at 4°C. Protein concentration was measured by the Bradford assay (Bio-Rad). Equal amounts of protein from nuclear extracts were resolved by 10% SDS-PAGE and then subjected to immunoblot analysis using anti-GAPDH antibody (GenScript USA Inc.; Cat #: A00192). Trypan blue exclusion test of cell viability HEK 293 cells were pretreated with the indicated concentrations of salicylates or glycyrrhizin for 1 h before MNNG treatment. For MNNG treatment, cells were incubated in media containing 100 μM MNNG for 1 h and then the MNNG-containing medium was replaced with fresh medium containing the indicated concentrations of salicylates or glycyrrhizin without MNNG. Floating as well as adherent cells were harvested 6 h after MNNG treatment by 0.05% Trypsin-EDTA (Corning) and centrifugation for 5 min at 100 x g and then the resulting cell pellets were resuspended in serum-free DMEM medium. To determine the cell viability, the cells were stained with a trypan blue staining solution (GIBCO) according to manufacturer’s protocol, which stains dead cells. Briefly, diluted cell suspension was mixed with an equal volume of 0.4% trypan blue solution and then the number of unstained (viable) and stained (nonviable) cells were counted using hemocytometer. 
 
 Discussion Several previous studies have demonstrated that high concentrations of salicylates (~10 mM) can induce apoptosis in a variety of cells [30–33]. For example, 10 mM sodium salicylate induced apoptosis in neutrophils by stimulating caspase-dependent turnover of Mcl-1, an anti-apoptotic member of the Bcl-2 family [33]. However, little was known about the effects of low concentrations of salicylate on apoptosis or cell death. Here we demonstrate that the low μM levels of SA and its natural and synthetic derivatives suppress cell death induced by MNNG, an agent which mimics neuronal cell death induced by ROS. This inhibition by these salicylates of cell death is likely due to their suppression of HsGAPDH nuclear translocation since it mirrors the effect of deprenyl. Deprenyl suppresses cell death by blocking HsGAPDH translocation to the nucleus [19]. Potentially therapeutic levels of SA for suppression of cell death are readily attainable since SA concentrations reaches ~140 μM (~20 mg/L) within 1 h following intake of 500 mg of aspirin [3]. Furthermore, the discovery of SA derivatives, which are 3–30 times more efficacious than SA, further supports the possibility that salicylates may be of therapeutic value in the treatment or even prevention of several widespread and devastating neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases. During the past half century, several targets of aspirin and its major metabolite SA have been identified. The most studied and celebrated are COX1 and COX2, whose enzymatic activities are irreversibly inhibited by aspirin’s acetylation of a serine in their catalytic site. This discovery by John Vane in the 1970’s [5] led to his selection for the Nobel Prize in Physiology or Medicine in 1982. More recently, additional potential targets have been uncovered, including Inhibitor of κB Kinase-β (IκK-β), whose kinase activity is inhibited by high concentrations of aspirin or SA [34]. IκK-β phosphorylates Inhibitor of κB, which leads to translocation of Nuclear Factor-κB (NF-κB) into the nucleus and the activation of NF-κB–regulated genes involved in inflammation, including cytokines. However, it is questionable whether IκK-β is a physiologically relevant target in vivo given that high concentrations of salicylates are needed to inhibit its kinase activity [35]. More recently, adenosine monophosphate-activated protein kinase (AMPK) was identified as an SA target [36]. High levels of salicylate (1 mM—30 mM) modestly increased the kinase activity of AMPK (1.5–2 fold) in vitro and in vivo. AMPK is a sensor of cellular energy levels, through which it regulates cell metabolism and growth; as such, it is involved in obesity and diabetes. In addition, supra-pharmacological concentrations SA (i.e., >5mM) have been shown to suppress phosphorylation, and hence inactivation, of retinoblastoma protein’s tumor suppression function, while inducing another tumor suppressor, p53 [35]. Using high-throughput screens that were developed to identify SABPs from plants [9–11], we have recently identified several novel SABPs from humans, including GAPDH and HMGB1, which was reported previously [25]. In contrast to all of the above human targets, SA suppresses the bioactivity of these two proteins in cells at low μM levels. Interestingly, these bioactivities are secondary, moonlighting functions of these two SABPs, some of which are shared by plant and animals. GAPDH in both plants and animals can be co-opted by some viruses to promote their replication, such as hepatitis A, B, C viruses in humans and tomato bushy stunt virus in plants [10,14–17]. Notably, SA interferes with the ability of plant GAPDH to bind the 3’ end of the minus strand of tomato bushy stunt viral RNA and thereby facilitate asymmetric replication [10]. Interestingly, Gly has anti-hepatitis virus activity and has been used in Japan for treatment of chronic hepatitis viral infection for decades [37–39]. Together, these observations suggest that SA or its more potent derivatives might also be useful for treatment of infection by this family of viruses. The discovery that 5-ASA binds HsGAPDH and inhibits its translocation to the nucleus suggests that this compound may modulate IBD through more than one mechanism. In addition to 5-ASA’s purported role in scavenging ROS produced by infiltrating, activated neutrophils, its ability to bind and suppress HsGAPDH translocation to the nucleus may block induction of cell death. Since several natural and synthetic SA derivatives exhibit even stronger binding to HsGAPDH and/or greater efficacy in suppressing nuclear translocation of HsGAPDH and cell death than 5-ASA, it is possible that they will not only have therapeutic value in the treatment of IBD, but that they will be even more efficacious. It is worth noting that 5-ASA’s therapeutic effect on IBD may also involve another human target of SA/aspirin, HMGB1 [25]. Dying cell release HMGB1, which has potent pro-inflammatory activities that can be inhibited by SA, ac3AESA or amorfrutin B1 [25]. While we have not tested 5-ASA’s ability to bind to and thereby inhibit HMGB1’s pro-inflammatory activities, it seems highly likely given 5-ASA’s chemical structure and the results of our structure–activity analyses with HsGAPDH and HMGB1. Although HsGAPDH binds 3AESA immobilized on a SPR sensor chip with high affinity (K d = 1.0 nmol/L), suppression of this interaction required millimolar concentrations of SA (Fig 2B). Suppression of 4AzSA crosslinking to HsGAPDH also required high levels of SA (Fig 1). Similar results were obtained with HMGB1, SA’s other newly-identified target [25]. These results suggest that both proteins have relatively low affinity for SA in vitro, despite inhibition of their bioactivities in vivo at low μmol concentrations. While these results might suggest that SA is acting through another molecule(s), which modulates the bioactivities of these two proteins, rather than directly via SA binding to HsGAPDH or to HMGB1, we strongly suspect that SA’s effect on HsGAPDH bioactivities is direct for the following reasons. First, we have rigorously established that HMGB1 is the direct target of SA using a mutant of HMGB1 (R24A/K28) whose SA-binding site was altered. This mutant protein retains its chemo-attractant activity, but this activity is no longer inhibitable by SA, because the mutant can no longer bind SA. Second, as with HMGB1, SA derivatives that bind to HsGAPDH more tightly than SA, also are stronger inhibitors of HsGAPDH nuclear translocation and cell death. Third, Gly, which appears to act as a mimic of SA both with HsGAPDH and with HMGB1, in which its binding site and that of SA overlap, has higher affinity for HsGAPDH than SA and is a stronger inhibitor of HsGAPDH nuclear translocation and cell death. Together the results with HsGAPDH (and with HMGB1) argue that SA’s effect on HsGAPDH nuclear translocation and cell death is due to its direct interaction with HsGAPDH. However, this has not been rigorously established using genetics. We suspect that in vivo the conformations of HsGAPDH (and of HMGB1) are modified by the interaction with other molecules, so that the binding of salicylates is facilitated. These “missing molecules” have yet to be identified. 
 
 Conclusion Taken together, the identification of HsGAPDH, as well as HMGB1, as targets for SA/aspirin in humans, coupled with a greater understanding of how salicylates regulate the activities of these proteins, has the potential to open broad new avenues in the treatment of a wide variety of diseases. Moreover, the identification of synthetic and natural derivatives of SA which are more potent inhibitors than SA of both HsGAPDH’s and HMGB1’s disease-associated bioactivities provides proof-of-concept that new SA-based molecules with high efficacy are attainable. 
 
 Supporting Information S1 Fig. Analysis of salicylic acid (SA)-binding activity of HsGAPDH by surface plasmon resonance. Sensorgrams of concentration-dependent HsGAPDH interacting with 3AESA immobilized on the SPR sensor chip. HsGAPDH has strong affinity for 3AESA with an apparent Kd of 1.01 nmol/L. https://doi.org/10.1371/journal.pone.0143447.s001 (TIF) 
 
 Acknowledgments We thank D’Maris Dempsy for assistance in editing the manuscript. 
 
 Author Contributions Conceived and designed the experiments: HWC MT MM MMH SWP DFK. Performed the experiments: HWC MT MM MMH SWP. Analyzed the data: HWC MT MM MMH SWP FCS DFK. Contributed reagents/materials/analysis tools: FCS. Wrote the paper: HWC MT DFK. Provided important insights and encouragement: SHS. 

 Aspirin and salicylic acid derivatives could help fight against a number of neurodegenerative diseases, according to new research published in PLOS One. 
 
 
 
 Aspirin: a drug of many talents. Could it be used to prevent neurodegenerative diseases? Aspirin: a drug of many talents. Could it be used to prevent neurodegenerative diseases? 
 
 Plants have been used for healing throughout history. Plant and herb-based medicine is still a primary form of treatment worldwide. 
 
 Around 50% of the pharmaceuticals developed over the past 20 years and approved by the US Food and Drug Administration (FDA) are either natural products or synthetic derivatives of natural products, mainly derived from plants. 
 
 Salicylic acid (SA) and its derivatives are a prime example. SA is the critical hormone for regulating the plant immune system. It is also the primary breakdown product of acetyl SA or aspirin. SA and its derivatives have been used for thousands of years to reduce pain, fever, and inflammation. 
 
 Americans alone consume 80 million aspirin tablets daily, and present-day targets of use include heart attack, stroke and certain cancers. 
 
 Senior author Daniel Klessig, a professor at Boyce Thompson Institute - Cornell University in Ithaca, NY, has studied the actions of SA for a long time, mostly focusing on plants. 
 
 Several targets in plants that are affected by SA have been identified in previous studies; many of these targets have equivalents in humans. 
 
 Role for aspirin in Alzheimer's and Parkinson's? 
 
 Now it seems that SA's impressive repertoire could expand to include Alzheimer's, Parkinson's and Huntington's disease; one of its components has been found to bind to an enzyme called Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), a culprit in these diseases. 
 
 GAPDH is a central enzyme in glucose metabolism but performs additional roles within the cell such as signaling. 
 
 Oxidative stress , which results from a lack of oxygen being delivered to the brain, is believed to contribute to these diseases. It leads to an excess of free radicals and other reactive compounds. 
 
 In these conditions, GAPDH is modified and enters the nucleus of neurons where it enhances protein turnover, leading to cell death. 
 
 The anti-Parkinson's drug deprenyl works by preventing GAPHD from entering into the nucleus. 
 
 Salicylic acid prevents GAPDH from entering nucleus 
 
 Researchers at the Boyce Thompson Institute and John Hopkins University - in Baltimore, MD - performed high-throughput screens to identify proteins in the human body that bind to SA. 
 
 They found that SA can bind to GAPDH and stop it from moving into the nucleus. They also found two other substances that bind more tightly to GAPDH than salicylic acid, in turn blocking GAPDH's movement into the nucleus and the resulting cell death more effectively. One is a natural derivative of salicylic acid from licorice, the other is a laboratory-synthesized derivative. 
 
 Earlier this year, Klessig's group identified another novel target of SA called High Mobility Group Box 1 (HMGB1), a protein that causes inflammation and is associated with several diseases, including arthritis, lupus, sepsis, atherosclerosis and certain cancers. 
 
 Low levels of SA block these pro-inflammatory activities, and the two other salicylic acid derivatives are 40-70 times more potent than salicylic acid at inhibiting them. 
 
 Klessig says: 
 
 "A better understanding of how salicylic acid and its derivatives regulate the activities of GAPDH and HMGB1, coupled with the discovery of much more potent synthetic and natural derivatives of salicylic acid, provide great promise for the development of new and better salicylic acid-based treatments of a wide variety of prevalent, devastating diseases." 
 
 Medical News Today recently reported on the benefits and risks of aspirin. 

 Aspirin targets key protein in neurodegenerative diseases 
 
 A new study finds that in cell cultures, a component of aspirin binds to an enzyme called GAPDH, which is believed to play a major role in neurodegenerative diseases, including Alzheimer’s, Parkinson’s and Huntington’s diseases. 
 
 Researchers at the Boyce Thompson Institute and John Hopkins University discovered that salicylic acid, the primary breakdown product of aspirin, binds to GAPDH, thereby stopping it from moving into a cell’s nucleus, where it can trigger the cell’s death. The study, which appears in the journal PLOS ONE, also suggests that derivatives of salicylic acid may hold promise for treating multiple neurodegenerative diseases. 
 
 Senior author Daniel Klessig, a professor at Boyce Thompson Institute and Cornell University, has studied the actions of salicylic acid for many years, but primarily in plants. Salicylic acid is the critical hormone for regulating the plant immune system. Previous studies have identified several targets in plants that are affected by salicylic acid, and many of these targets have equivalents in humans. 
 
 In the new study, the researchers performed high-throughput screens to identify proteins in the human body that bind to salicylic acid. GAPDH, (Glyceraldehyde 3-Phosphate Dehydrogenase), is a central enzyme in glucose metabolism, but plays additional roles in the cell. Under oxidative stress—an excess of free radicals and other reactive compounds—GAPDH is modified and then enters the nucleus of neurons, where it enhances protein turnover, leading to cell death. 
 
 The anti-Parkinson’s drug deprenyl blocks GAPDH’s entry into the nucleus and the resulting cell death. The researchers discovered that salicylic acid also is effective at stopping GAPDH from moving into the nucleus and preventing cell death. 
 
 “The enzyme GAPDH, long thought to function solely in glucose metabolism, is now known to participate in intracellular signaling,” said co-author Solomon Snyder, professor of neuroscience at Johns Hopkins University in Baltimore. “The new study establishes that GAPDH is a target for salicylate drugs related to aspirin, and hence may be relevant to the therapeutic actions of such drugs.” 
 
 Furthermore, they found that a natural derivative of salicylic acid from the Chinese medical herb licorice and a lab-synthesized derivative bind to GAPDH more tightly than salicylic acid. Both are more effective than salicylic acid at blocking GAPDH’s movement into the nucleus and the associated cell death. 
 
 Earlier this year, Klessig’s group identified another novel target of salicylic acid called HMGB1 (High Mobility Group Box 1), which causes inflammation and is associated with several diseases, including arthritis, lupus, sepsis, atherosclerosis and certain cancers. Low levels of salicylic acid block these pro-inflammatory activities, and the above mentioned salicylic acid derivatives are 40 to 70 times more potent than salicylic acid at inhibiting these pro-inflammatory activities. 
 
 “A better understanding of how salicylic acid and its derivatives regulate the activities of GAPDH and HMGB1, coupled with the discovery of much more potent synthetic and natural derivatives of salicylic acid, provide great promise for the development of new and better salicylic acid-based treatments of a wide variety of prevalent, devastating diseases,” said Klessig.
– Aspirin is already used for pain relief and as a treatment for cardiovascular disease and even cancer, Medical News Today reports—and now a new study published in PLOS One finds it could also be useful in the fight against Alzheimer's and other neurodegenerative diseases. The central factor in the new findings: salicylic acid, the "primary breakdown product of aspirin," per a press release. An enzyme called GAPDH, if put under stress due to a lack of oxygen being delivered to the brain, can enter the nucleus of neurons, and once there it leads to cell death. GAPDH is believed to play a big role in diseases including Alzheimer's, Parkinson's, and Huntington's, and one anti-Parkinson's drug blocks the enzyme from entering the nucleus. The new study finds that salicylic acid binds to GAPDH and also bars it from entering. Further research found that both a lab-synthesized derivative and a natural derivative of salicylic acid (the natural derivative comes from the herb licorice) bind to GAPDH even more tightly, and do an even better job at blocking its entry into the nucleus and causing cell death. As such, "salicylates may be of therapeutic value in the treatment or even prevention of several widespread and devastating neurodegenerative diseases," the study states. Adds the senior author, "A better understanding of how salicylic acid and its derivatives regulate the activities of GAPDH ... coupled with the discovery of much more potent synthetic and natural derivatives of salicylic acid, provide great promise for the development of new and better salicylic acid-based treatments of a wide variety of prevalent, devastating diseases." (If you're over 50, here's why you should take aspirin.)