What is carbonic anhydrase?

Novel drug discovery and development play a crucial role in addressing diseases like cancer. One promising target for anticancer drug development is carbonic anhydrase (CA). But why focus on this enzyme?

Carbonic anhydrases are zinc metalloprotein enzymes that catalyze the conversion of carbon dioxide (CO₂) into bicarbonate while releasing a proton. These enzymes facilitate the transport of CO₂ and protons across biological membranes, affecting intercellular, intracellular, and extracellular regions. They play essential roles in regulating pH, transporting ions, and contributing to processes such as bone resorption and the secretion of gastric, cerebrospinal fluid, and pancreatic juices.

The human body expresses 16 isoforms of the carbonic anhydrase family, including CAIX and CAXII.¹ The carbonic anhydrases found in human cells are referred to as human carbonic anhydrases (hCAs).

Read on to gain further insights into the role of this enzyme in cancer treatment.

Physiological function of hCAs

Human carbonic anhydrases (hCAs) exhibit catalytic activity in two stages. In the first stage, the hydroxide ion, which is linked to the metal ion, performs a nucleophilic attack on the CO₂ molecule, leading to the formation of bicarbonate. In the second stage, water molecules coordinate with the metal ions, displacing bicarbonate.

This two-step mechanism of carbonic anhydrases is crucial for various physiological and pathological processes, including:²

• Transport of CO₂ and bicarbonate
• Regulation of pH homeostasis
• Electrolyte secretion
• Metabolic pathways such as gluconeogenesis, lipogenesis, and ureagenesis
• Calcification and bone resorption
• Maintenance of osmotic pressure
• Transport of calcium and sodium
• Neuronal signalling
• Tumorigenicity

Mechanism of CA in cancer cells

Metastatic tumours—cancer cells that break away from the primary tumour and spread to other parts of the body—create a hypoxic extracellular environment due to ongoing cell proliferation.³ This hypoxic condition arises from the expansion of blood supply in tumour cells, resulting in low oxygen concentrations within the tumour. Hypoxia leads to extracellular acidosis, promoting the use of glycolytic metabolites that produce lactic acid and lower the pH of the tumour microenvironment.

Carbonic Anhydrases IX (CAIX) and XII (CAXII) play a crucial role in regulating the pH of the tumour microenvironment, promoting tumour cell survival under these extreme conditions. These enzymes facilitate the transfer of bicarbonate ions from reversible CO₂ hydration into cells through anion exchangers and Na⁺/HCO₃⁻ co-transporters, helping to maintain intracellular pH. The overexpression of CAIX and CAXII contributes to tumour growth, activates the metastatic cascade, and reduces responsiveness to chemotherapies.³ 

By aiding cancer cells in surviving under stressful conditions, targeting these carbonic anhydrases becomes an important strategy for extending the lives of cancer patients.

Targeting CA IX and CA XII

Among the 15 carbonic anhydrase isoforms expressed in humans, only CAIX and CAXII have been linked to cancer.^{3, 4} These enzymes play a crucial role in regulating the tumour microenvironment. CAIX, in particular, is significant due to its high expression in solid tumours and low expression in normal tissues.

Inhibiting the activity of either CAIX or CAXII can impact the pH of the tumour microenvironment, thereby limiting tumour cell survival and proliferation. These characteristics make CAIX and CAXII promising targets for anti-cancer therapies. While other isoforms may also have potential for cancer targeting, their specific functions remain largely unknown despite being expressed and upregulated in tumours.⁴

Research has suggested that CAIX may contribute to resistance against radiation and chemotherapy, indicating that blocking this enzyme could enhance treatment effectiveness. Studies suggest that modifications to the tumour microenvironment may explain the increased anticancer impact of combination therapies involving radiation and sulfonamide-bearing inhibitors.

The enzyme's critical role in pH regulation is believed to mediate the anticancer effects of CAIX inhibition. Furthermore, recent findings indicate that CAIX interacts with various signalling pathways involved in the radiation-induced cellular response. Inhibiting CAIX activity in hypoxic cancer cells could significantly enhance the ability to specifically target these cells while minimizing damage to normal tissues.^1,4,5

Synthetic CA inhibitors

Numerous compounds have been developed to inhibit various isoforms of carbonic anhydrase. Clinically available carbonic anhydrase inhibitors include:⁵

• Acetazolamide (AZA)
• Methazolamide
• Dorzolamide
• Brinzolamide
• Diclofenamide
• Ethoxazolamide
• Zonisamide, and 
• Indisulam

These inhibitors are used to treat a variety of conditions, including ocular hypertension, glaucoma, oedema, and epilepsy.⁵ However, acetazolamide is a non-selective carbonic anhydrase inhibitor that strongly inhibits most human CA isoforms, making it unsuitable for developing isoform-selective drugs.

To address this limitation, a "tail approach" has been employed to create inhibitors that are selective for CAIX and CAXII. The concept is straightforward: attach moieties that impart the desired characteristics to enhance selectivity.

Clinical application: how carbonic anhydrase inhibitors are administered

Clinically available carbonic anhydrase inhibitors (CAIs) can be administered in various forms, including oral tablets and topical preparations, depending on the specific medication and the condition being treated.⁶ Acetazolamide, the prototypical CAI, is commonly administered in tablet form to treat conditions such as glaucoma, altitude sickness, and certain types of oedema.⁵

In contrast, dorzolamide and brinzolamide are available as topical eye drops specifically designed for managing intraocular pressure in glaucoma patients. These formulations leverage the inhibition of carbonic anhydrase to decrease aqueous humour production, thereby lowering the pressure within the eye​.⁵ 

This versatility in administration allows for targeted treatment options tailored to the patient's needs and the nature of the condition.

Summary

Carbonic anhydrases are a class of enzymes expressed in human cells that perform various functions. Certain isoforms of these enzymes are overexpressed in specific conditions, such as cancer, where they assist cancer cells in survival. This overexpression makes these enzymes novel targets for drug development aimed at enhancing cancer treatment outcomes and improving patient quality of life.

CAIX and CAXII are particularly targeted, as research indicates high levels of activity in cancer cells. Thus, carbonic anhydrase inhibitors are poised to become important components of future anticancer therapies, improving treatment for cancer patients.