Aspirin could act as a suicide inhibitor of carbonic anhydrase II

aliml

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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.
[...]

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.



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:
 
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aliml

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Platelet Carbonic Anhydrase II, a Forgotten Enzyme, May Be Responsible for Aspirin Resistance​

Background​

Thromboembolic events constitute a major health problem, despite the steadily expanding arsenal of antiplatelet drugs. Hence, there is still a need to optimize the antiplatelet therapy.

Objectives​

The aim of our study was to verify a hypothesis that there are no differences in platelet proteome between two groups of healthy people representing different acetylsalicylic acid (aspirin) responses as assessed by the liquid chromatography/mass spectrometry (LC/MS) technique.

Patients/Methods​

A total of 61 healthy volunteers were recruited for the study. Physical examination and blood collection were followed by platelet-rich plasma aggregation assays and platelet separation for proteomic LC/MS analysis. Arachidonic acid- (AA-) induced aggregation (in the presence of aspirin) allowed to divide study participants into two groups aspirin-resistant (AR) and aspirin-sensitive (AS) ones. Subsequently, platelet proteome was compared in groups using the LC/MS analysis.

Results​

The LC/MS analysis of platelet proteome between groups revealed that out of all identified proteins, the only discriminatory protein, affecting aspirin responsiveness, is platelet carbonic anhydrase II (CA II).

Conclusions​

Carbonic anhydrase II (CA II) is a modulator of platelet function. Increased activity and/or concentration of CA II in platelets should be rated as a new independent risk factor for aspirin resistance and thus for thromboembolic events. There may be a need to use the drugs inhibiting CA in clinical setting more often nowadays, especially in patients with increased platelet activity/amount of CA II.
 

Mauritio

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Very interesting. Thanks for posting! Another win for aspirin.

So if I'm reading this right, aspirin (precise:salicylic acid) forms an unbreakable bond with carbonic anhydrase II .
I dont see a reason why this wouldn't take place independent of the dosage. Of course the reaction would increase with an increasing blood concentration of aspirin.

What I'm wondering is what amount of aspirin is needed to achieve an effect that's comparable with what acetazolamide does. I wouldn't be surprised if it was in the 1g+ territory and part of its uncoupling effects stem from increased CO2 through CA-Inhibition.


Ray wrote about this 10 years ago already :
"Existing carbonic anhydrase inhibitors, such as
acetazolamide, will inhibit those enzymes, without harming other tissues. Aspirin has some effect as an inhibitor of carbonic anhydrase (Bayram, et al.,2008)."

From the study ray cited:
IC50:
Salicylic acid: 0.56 mM (CA1); 0.68 (CA2)
Aspirin: 2.71 mM (CA1); 1.16 (CA2)
(In vitro inhibition of salicylic acid derivatives on human cytosolic carbonic anhydrase isozymes I and II - PubMed)


I dont know how to calculate the dosage for that.
But haidut said this in another thread:

"...oral doses of aspirin needed to achieve the optimal concentration of 0.5mM (as shown on the image and other studies) are in the range of 1,000mg-1,500mg as a loading first dose, followed by 500mg every 4-6 hours."

So by extrapolating the dosage from this quote we would need an aspirin dose in the range of 5g+ to significantly inhibit carbonic anhydrase 1 !

The other value for salicylic acid is indeed in the 1g+ territory, so quite achievable for a human. And aspirin gets rapidly converted to salicylic acid inside the body so I would leant towards using this value as a guide!

So unsurprisingly the uncoupling dose matches the carbonic anhydrase-inhibting dose!
Makes a good case for at least semi-regular dosing of 1g+ of aspirin.
 
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Mauritio

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The study ray cited mentions a few interesting substances that inhibit carbonic anhydrase 1+2 at much lower concentrations.
One substance is the compound named 4-aminosalicylic, also known as Para-Aminosalicylic Acid.

It has a much lower IC50 for carbonic anhydrase(0.13mMol)so a much lower dosage might be sufficient.

Don't know if anybody has ever tried it, but it seems to be used in children and adults in dosages that are much higher than what would be needed to inhibit carbonic anhydrase.
 
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aliml

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From the study ray cited:
IC50:
Salicylic acid: 0.56 mM (CA1); 0.68 (CA2)
Aspirin: 2.71 mM (CA1); 1.16 (CA2)
(In vitro inhibition of salicylic acid derivatives on human cytosolic carbonic anhydrase isozymes I and II - PubMed)

I dont know how to calculate the dosage for that.
Thanks, I took help from the explanation in the link below to calculate the dosage!
The molar mass of Aspirin is 180.158 g/mol (0.180158 g).
So.
180.158 g/mol * 2.71 mmol/L == 488 mg
180.158 g/mol * 1.16 mmol/L == 209 mg

1658217212167.png

Higher doses of aspirin are required for complete inhibition.
180.158 g/mol * 7.53 mmol/L == 1357 mg
180.158 g/mol * 3.66 mmol/L == 660 mg

1658217065801.png

I hope these calculations are correct!
 

Mauritio

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Thanks, I took help from the explanation in the link below to calculate the dosage!
The molar mass of Aspirin is 180.158 g/mol (0.180158 g).
So.
180.158 g/mol * 2.71 mmol/L == 488 mg
180.158 g/mol * 1.16 mmol/L == 209 mg

View attachment 39131

Higher doses of aspirin are required for complete inhibition.
180.158 g/mol * 7.53 mmol/L == 1357 mg
180.158 g/mol * 3.66 mmol/L == 660 mg

View attachment 39128

I hope these calculations are correct!
Very interesting! Thanks.

Although I'm not sure if its correct because then haiduts comment wouldnt make much sense.
To achieve the 0.5mM you'd only need about 100mg. He talked about that dose in the context of uncoupling and I doubt you'd get any significant uncoupling through 100mg of aspirin.
Could it somehow be that you're off by a decimal?
 
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aliml

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Although I'm not sure if its correct because then haiduts comment wouldnt make much sense.
To achieve the 0.5mM you'd only need about 100mg. He talked about that dose in the context of uncoupling and I doubt you'd get any significant uncoupling through 100mg of aspirin.
Could it somehow be that you're off by a decimal?
Maybe this calculation is correct!
The median serum concentration of salicylic acid in patients taking aspirin (75 mg daily) was 10.03 µmol/litre (0.01003 mmol/l).
Salicylic acid:
CA1: (0.56 mmol/l * 75 mg) / 0.01003 mmol/l == 4187 mg aspirin
CA2: (0.68 mmol/l * 75 mg) / 0.01003 mmol/l == 5084 mg aspirin
 

Mauritio

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I'm not sure tbh. Maybe @haidut or someone else can chime in on the dosage.
 

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