Abstract
Twelve carbonic anhydrase (CA) isoforms catalyze carbon dioxide hydration to bicarbonate and acid protons and are responsible for many biological functions in human body. Despite their vital functions, they are also responsible for, or implicated in, numerous ailments and diseases such as glaucoma, high altitude sickness, and cancer. Because CA isoforms are highly homologous, clinical drugs designed to inhibit enzymatic activity of a particular isoform, can also bind to others with similar affinity causing toxic side effects. In this study, the affinities of twelve CA isoforms have been determined for nineteen clinically used drugs used to treat hypertension related diseases, i.e. thiazides, indapamide, and metolazone. Their affinities were determined using a fluorescent thermal shift assay. Stopped flow assay and isothermal titration calorimetry were also employed on a subset of compounds and proteins to confirm inhibition of CA enzymatic activity and verify the quantitative agreement between different assays. The findings of this study showed that pharmaceuticals could bind to human CA isoforms with variable affinities and inhibit their catalytic activity, even though the drug was intended to interact with a different (non-CA) protein target. Relatively minor structural changes of the compounds may cause significant changes in affinity and selectivity for a particular CA isoform.
Highlights
Pharmaceuticals are designed to bind target proteins selectively; in practice, they often bind to other non-intended targetscausing undesired side effects
Compounds were obtained from the following sources: 2-chlorobenzenesulfonamide, althiazide, bendroflumethiazide, benzthiazide, chlorothiazide, clopamide, cyclothiazide, dichlorofenamide, hydroflumethiazide, indapamide, metalozone, polythiazide, trichlormethiazide and xipamide were obtained from Sigma-Aldrich; chlorthalidone, clorexolone, fenquizone, and quinethazone were obtained from SantaCruz Biotechnology; hydrochlorothiazide and furosemide were obtained from AlfaAesar
Compound 1, 2-chloro-benzenesulfonamide, is a fragment of the remaining compounds, all of which have been used in the clinic
Summary
Pharmaceuticals are designed to bind target proteins selectively; in practice, they often bind to other non-intended targetscausing undesired side effects. While the mechanism of lowering blood pressure is still poorly understood, the most likely targets are ion transporters [1, 2], ion channels, carbonic anhydrases (CA), and other proteins could play a role [1, 2]. The inhibition of CA by some members of this compound class has been demonstrated [3].
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