Aspirin: A Suicide Inhibitor of Carbonic Anhydrase II
Carbonic anhydrase II (CAII) is a metalloenzyme that catalyzes the reversible hydration/dehydration of CO[2] /HCO[3] [−] . In addition, CAII is attributed to other catalytic reactions, including esterase activity. Aspirin (acetyl-salicylic acid), ...
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Abstract
Carbonic anhydrase II (CAII) is a metalloenzyme that catalyzes the reversible hydration/dehydration of CO2/HCO3−. In addition, CAII is attributed to other catalytic reactions, including esterase activity. Aspirin (acetyl-salicylic acid), an everyday over-the-counter drug, has both ester and carboxylic acid moieties. Recently, compounds with a carboxylic acid group have been shown to inhibit CAII. Hence, we hypothesized that Aspirin could act as a substrate for esterase activity, and the product salicylic acid (SA), an inhibitor of CAII. Here, we present the crystal structure of CAII in complex with SA, a product of CAII crystals pre-soaked with Aspirin, to 1.35Å resolution. In addition, we provide kinetic data to support the observation that CAII converts Aspirin to its deacetylated form, SA. This data may also explain the short half-life of Aspirin, with CAII so abundant in blood, and that Aspirin could act as a suicide inhibitor of CAII.Introduction
Carbonic anhydrases (CAs) are a family of mainly zinc metalloenzymes responsible for the interconversion of carbon dioxide (CO2) into bicarbonate (HCO3−) and a proton via a ping-pong mechanism [Equation (1)] [1]. As such, CAs play an important role in blood homeostasis, CO2/HCO3− transportation, and pH regulation [2]. There are 12 catalytic isoforms of CA expressed in humans, each with unique amino acid sequences, catalytic rates, cellular location, and tissue expression [2]. The active site of human CAs is conserved, with a zinc ion coordinated by three histidine (H94, H96, and H119 (CAII numbering)) and a water/hydroxide [3]. Of these isoforms, CAII is the most widely expressed isoform, responsible for regulating the intracellular pH in nearly every cell [4]. CAII is the fastest human CA, with a kcat of ~1100ms−1 that approaches the rate of diffusion [5].CAs play a critical role in physiology, to increase the rate of CO2/HCO3− interconversion (Equation (1)) [4]. HCO3− is the most commonly transported form of CO2 in the body [4]. Large quantities of CO2 are produced in tissues during respiration before removal by red blood cells (RBC) and transported to the lungs [4]. While CAII plays a large role in transporting CO2, it isn’t the only mode of excretion. CAII expression levels are elevated in the kidney as it regulates HCO3− flux [6]. CAII also balances cytoplasmic pH via interactions with a variety of membrane-bound ion carriers, including MCT1 and 4 [4].
In addition, CAII is important in blood homeostasis [4]. Human RBCs contain a high concentration of CAII at 0.8 attomol [7]. CAII has also been shown to be involved in regulating platelet function. While the exact mechanism is unknown, CAII is known to be involved in nitrocysteine and nitric oxide formation, both critical in platelet inhibition [8].
As CAs are responsible for a variety of physiological functions and pH regulation, they are often clinically targeted. CA inhibitors (CAIs) are used to treat a variety of diseases such as glaucoma, altitude sickness, and epilepsy [9]. In addition, CAIs are currently being developed as anti-cancer drugs [10,11,12]. These inhibitors are designed to bind to the active site zinc, displacing the zinc bound solvent. The most common type of CAIs are sulfonamides, such as acetazolamide, which has nM binding affinity. Many of these sulfonamide-based molecules are used clinically, such as dorzolamide, for the treatment of glaucoma [13,14]. In addition to sulfonamides, a variety of other chemical motifs have been identified to inhibit CA, such as carboxylic acids [15]. Nicotinic and ferulic acid have recently been identified as inhibitors of CAII [16]. Unlike the sulfonamide-based drugs, these inhibitors do not directly displace the zinc bound solvent, but instead anchor through the solvent, blocking substrate entry to the active site [16]. Furthermore, 3-nitrobenzoic acid has also been reported as a potent CAI, with further studies showing its potential clinical relevance as a cancer therapeutic [17]. Previous work has also shown that salicylic acid as well as some phenol derivatives are μM inhibitors of mammalian CAs, although the exact mechanism of inhibition for salicylic acid is unknown [18]. These carboxylic acid-based compounds represent a new and largely unstudied class of CAIs.
Aspirin (acetylsalicylic acid) is one of the most widely studied and consumed drugs in use. Aspirin is a known cyclooxygenase (COX) inhibitor, giving the molecule its anti-inflammatory and blood thinning characteristics [19]. Aspirin inhibits the COX enzymes by acetylating critical active site residues, leaving the enzymes acatalytic while generating salicylic acid (SA) as a byproduct [19]. While Aspirin is typically used by patients prone to heart disease, there are many hypotheses about its other potential therapeutic benefits, such as a chemotherapy or a preventive of preeclampsia [19,20,21]. Each year, 4 × 104 metric tons of Aspirin are consumed, which equate to ~120 billion pills [21]. A typical dose of Aspirin is 325 mg; however, there are lower dosage options for everyday use and higher concentrations (up to 6 g per day, or ~7 mM in blood) for at-risk patients with heart disease [22]. Interestingly, Aspirin only has a half-life of ~15 min in blood due to a previously unidentified carboxylesterase [23]. The short half-life of Aspirin leads to patients taking the drug daily to keep a therapeutic dose in their system. A recent study found in a genome-wide search that CAII is the only protein overexpressed in patients with Aspirin resistance and therefore may be the unidentified carboxylesterase [24]. Since Aspirin is a carboxylic acid-based molecule, it was hypothesized that it could potentially bind to CAII. Here, we examine this hypothesis through structure activity relationship studies between Aspirin and CAII, through X-ray crystallography and a spectroscopy-based kinetic assay. We determine that CAII is the previously unidentified carboxylesterase responsible for Aspirin’s short half-life in the blood, and that the product of this reaction, SA, can then inhibit CAII, thus making Aspirin a suicide inhibitor.
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Conclusions
Based on these findings, we conclude that Aspirin binds and inhibits CAII via the SA product, as it retains the carboxylic acid motif similar to other CAIs such as nicotinic, ferulic, and 3-nitrobenzoic acids. These findings imply CAII’s importance in the blood, beyond its carbonic anhydrase activity and further implicate the enzyme in platelet function. We have identified CAII as the carboxylesterase responsible for Aspirin’s short half-life in the blood. Therefore, perhaps a combined therapy with a CA inhibitor and Aspirin could improve Aspirin’s efficacy in the treatment of heart disease.Suicide inhibition - Wikipedia
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In biochemistry, suicide inhibition, also known as suicide inactivation or mechanism-based inhibition, is an irreversible form of enzyme inhibition that occurs when an enzyme binds a substrate analog and forms an irreversible complex with it through a covalent bond during the normal catalysis reaction. The inhibitor binds to the active site where it is modified by the enzyme to produce a reactive group that reacts irreversibly to form a stable inhibitor-enzyme complex. This usually uses a prosthetic group or a coenzyme, forming electrophilic alpha and beta unsaturated carbonyl compounds and imines.
Some clinical examples of suicide inhibitors include:
- Disulfiram, which inhibits the acetaldehyde dehydrogenase enzyme.
- Aspirin, which inhibits cyclooxygenase 1 and 2 enzymes.
- Clavulanic acid, which inhibits β-lactamase: clavulanic acid covalently bonds to a serine residue in the active site of the β-lactamase, restructuring the clavulanic acid molecule, creating a much more reactive species that attacks another amino acid in the active site, permanently inactivating it, and thus inactivating the enzyme β-lactamase.
- Penicillin, which inhibits DD-transpeptidase from building bacterial cell walls.
- Sulbactam, which prohibits penicillin-resistant strains of bacteria from metabolizing penicillin.
- AZT (zidovudine) and other chain-terminating nucleoside analogues used to inhibit HIV-1 reverse transcriptase in the treatment of HIV/AIDS.
- Eflornithine, one of the drugs used to treat sleeping sickness, is a suicide inhibitor of ornithine decarboxylase.
- Nerve agent and related pesticides such as parathion are organophosphorus suicide inhibitors of acetylcholinesterase with aging times dependent on the lability of leaving groups present on the organophosphorus moiety of the molecule.[1]
- 5-fluorouracil acts as a suicide inhibitor of thymidylate synthase during the synthesis of thymine from uridine. This reaction is crucial for the proliferation of cells, particularly those that are rapidly proliferating (such as fast-growing death cancer tumors). By inhibiting this step, cells die from a thymineless death because they have no thymine to create more DNA. This is often used in combination with methotrexate, a potent inhibitor of dihydrofolate reductase enzyme.
- O6-Benzylguanine, a drug which depletes O6-alkylguanine-DNA alkyltransferase by virtue of its similarity to the DNA repair protein's target lesion.
- Exemestane, a drug used in the treatment of breast cancer, is an inhibitor of the aromatase enzyme.
- Selegiline,[2] although in the attached reference the compound is called a 'suicide inactivator' (not inhibitor).
- Vigabatrin, an anticonvulsant, is a suicide inhibitor of GABA-T.