Unveiling the Carbonic Anhydrase Inhibitory Profile of Aprocitentan: Kinetic and Structural Characterization

Abstract

Aprocitentan, a dual endothelin receptor antagonist recently approved for the treatment of resistant hypertension, contains a sulfamide moiety structurally related to known zinc-binding groups found in carbonic anhydrase (CA) inhibitors. Given the central role of CAs in renal and vascular physiology, we investigated whether aprocitentan exerts secondary pharmacological effects via CA inhibition. Kinetic profiling of aprocitentan was performed against all catalytically active human CA isoforms (hCA I–XIV). In addition, X-ray crystallographic structures of aprocitentan in complex with hCA I and hCA II were solved to elucidate the binding mode at the molecular level. These findings highlight a previously unrecognized dual mechanism of action for aprocitentan involving not only endothelin receptor antagonism but also CA inhibition. The combined effect may enhance its antihypertensive efficacy and suggests new avenues for therapeutic exploration in resistant hypertension.

Hypertension is a leading modifiable risk factor contributing to cardiovascular diseases. Globally, it affects approximately 1.28 billion adults, with nearly 46% unaware of their condition. The pathophysiological mechanisms of hypertension are multifactorial, involving complex interactions among blood volume regulation, vascular tone, and endothelial function. Disruption in any of these physiological components can result in elevated blood pressure, ultimately causing organ damage and increasing the risk of cardiovascular complications. , Several classes of medications are commonly used as first-line treatments for hypertension. These include angiotensin II receptor blockers, angiotensin-converting enzyme inhibitors, dihydropyridine calcium channel blockers, and thiazide diuretics, initially promoting the excretion of sodium and chloride in the nephron, thereby reducing the extracellular fluid volume. With long-term use, they also contribute to decreased peripheral vascular resistance. , Despite the availability of these therapies, more than 10% of patients with hypertension do not achieve adequate blood pressure control even when treated with a combination of three antihypertensive agents from different classes (typically a diuretic, a renin–angiotensin system inhibitor, and a long-acting calcium channel blocker) at their maximum tolerated doses. , This clinical scenario is called resistant hypertension. This condition significantly increases the risk of organ damage, including hypertensive retinopathy, carotid intima–media thickening, left ventricular hypertrophy, myocardial infarction, stroke, heart failure, and progressive renal impairment.

In response to this growing clinical challenge, in 2024 both the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) approved aprocitentan (Figure ), a novel first-in-class dual endothelin receptor antagonist that selectively targets both endothelin receptor type A (ET_A) and endothelin receptor type B (ET_B) receptors with a binding selectivity ratio of approximately 1:16. Both are G-protein-coupled receptors found on cells, and they play a role in various physiological and pathophysiological processes, particularly in the cardiovascular system and pulmonary hypertension. Aprocitentan is indicated for the management of resistant hypertension in patients who are already receiving at least three antihypertensive agents. , This approval marks a significant advancement in the therapeutic landscape, offering a new and promising mechanism of action to address the difficult-to-treat patient population. Over the past decades, only three endothelin receptor antagonists, namely, bosentan, ambrisentan, and macitentan (Figure ), have been approved for clinical use, primarily for the treatment of pulmonary arterial hypertension. −

1.

ET_A and ET_B receptor antagonists in clinical use.

Aprocitentan, the major active metabolite of macitentan, belongs to the sulfamide class. This class of compounds are considered bioisosteres of sulfonamides, a well-known zinc binding group present in carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs). − CAs are a family of zinc-containing metalloenzymes that catalyze the reversible conversion of carbon dioxide to bicarbonate and a proton. − This reaction plays a fundamental role in maintaining acid–base balance, regulating electrolyte secretion, facilitating ion transport, and supporting various biosynthetic processes. −

Although newer-generation diuretics, including loop diuretics, thiazides, thiazide-like agents, and aldosterone antagonists, are now more commonly employed in clinical practice, many of these drugs still retain varying degrees of CA inhibitory activity. Notably, acetazolamide (AAZ), a prototypical CAI, continues to be used in the treatment of idiopathic intracranial hypertension, acute mountain sickness, and certain types of glaucoma. Beyond their renal effects, CAs and their inhibitors are increasingly recognized for their involvement in vascular physiology. Several CA isoforms are expressed in vascular smooth muscle cells and endothelial cells, where they are thought to contribute to vessel tone regulation and local blood flow control. This suggests a potential role for CA activity, either directly or indirectly, in modulating vascular function and hemodynamics. − Given this broader physiological significance, we investigated whether aprocitentan is able to inhibit all catalytically active human CA isoforms (hCA I–XIV), potentially contributing to its observed reduction of hypertension. In addition, we determined the X-ray crystal structures of aprocitentan bound to hCA I and hCA II, providing structural insights into its binding interactions and inhibition mechanism. The inhibition profiles of aprocitentan and AAZ, a clinical sulfonamide CAI used as a reference, are presented in Table .

1. Inhibition Data of All Human CA Isoforms with Aprocitentan and AAZ Collected by a Stopped-Flow CO₂ Hydrase Assay.

	K I (μM)
isoform	aprocitentan	AAZ
hCA I	21.5	0.250
hCA II	5.98	0.012
hCA III	>100	>100
hCA IV	>100	0.074
hCA VA	33.8	0.063
hCA VB	30.5	0.054
hCA VI (salivary)	5.25	0.011
hCA VII	0.61	0.002
hCA IX	0.69	0.026
hCA XII	2.84	0.006
hCA XIII	1.78	0.017
hCA XIV	3.49	0.041

^aMeans of three different assays by a stopped flow technique (errors were in the range of ±5–10% of the reported values).

Aprocitentan showed a markedly different inhibition profile compared to AAZ, especially toward the ubiquitous cytosolic isoforms hCA I and hCA II. Indeed, aprocitentan was approximately 100-fold less potent than AAZ against hCA I and nearly 500-fold less potent against hCA II, with inhibition constant (K _I) values of 21.5 and 5.98 μM, respectively. The isoform hCA III was not inhibited by either aprocitentan and AAZ (K _I > 100 μM). Similarly, hCA IV was not inhibited when aprocitentan was tested. The mitochondrial isoforms VA and VB were weakly inhibited by aprocitentan, with K _I values of 33.8 and 30.5 μM, respectively, and no significant selectivity was observed between the two isoforms. Interestingly, hCA VII and the tumor-associated hCA IX emerged as the isoforms most inhibited by aprocitentan, with nanomolar K _I values of 0.61 and 0.69 μM, respectively. In contrast, hCA XII was approximately 4 times less inhibited than hCA IX. Finally, aprocitentan showed moderate inhibitory activity against hCA XIII (K _I = 1.78 μM) and hCA XIV (K _I = 3.49 μM).

Given the high expression levels of the cytosolic isoforms hCA I and hCA II in kidney tissue and hCA II in the endothelium of blood vessels, which elaborates numerous vasoconstricting and vasodilating substances, we investigated the key interactions of aprocitentan with these isoforms by crystallographic analysis and successfully solved the X-ray crystal structures of their complexes. This structural insight aimed to elucidate the lower inhibitory activity against these two isoforms compared to classical sulfamide compounds. Indeed, previous studies reported that sulfamide inhibitors act as potent inhibitors in the nanomolar range, especially for hCA II. ,

Examination of the electron density map for the hCA II–aprocitentan complex revealed well-defined electron density in the active site, fully compatible with the structure of the sulfamide inhibitor (Figure S1). The sulfamide moiety coordinates the catalytic Zn­(II) ion through its deprotonated nitrogen, mirroring the binding mode typically observed with classical sulfonamide-based CAIs. , A conserved feature shared with sulfonamides is the interaction between the sulfamide nitrogen and Thr199 as well as the hydrogen bond between one of the sulfamide oxygen atoms and the backbone of Thr199, which stabilizes the complex. , However, in contrast to sulfonamides but consistent with other sulfamide inhibitors, a second hydrogen bond is formed between the additional nitrogen atom of the sulfamide group and Thr200. , This structural evidence suggests that the relatively weaker inhibition observed is not due to the sulfamide zinc-binding group itself but rather to the bulky ortho-substituted pyrimidine ring. Indeed, the 4-bromophenyl moiety is accommodated in the hydrophobic pocket of hCA II, engaging in van der Waals interactions with Phe131 and Leu198. These interactions appear to shift the pyrimidine core into an unusual region of the active site delimited by Asn62, Asn67, and Gln92, a site typically occupied by the cryoprotectant glycerol, employed in crystallographic experiments. This orientation was not commonly observed for sulfamide-based inhibitors (Figure ). This unfavorable positioning likely contributes to the reduced inhibitory potency toward hCA II. On the other hand, the second pyrimidine ring, located in in the tail of aprocitentan, was located in the hydrophilic pocket of the enzyme active site, facilitating multiple hydrogen bonds with Asn67 and Gln92 and an additional water bridge with Glu69, enhancing local stabilization of this moiety (Figure ).

2.

X-ray crystal structure of hCA II with bound aprocitentan (PDB ID 9RUZ). Residues involved in binding of the inhibitor are also shown. The gray sphere represents the zinc ion in the active site of the protein. Purple dotted lines show the coordination of the zinc ion; van der Waals interactions are shown in blue, and hydrogen bonds and water bridges are shown in red.

We next investigated the binding mode of aprocitentan in complex with hCA I, making the first structural report of a sulfamide-based inhibitor bound to this isoform. As with hCA II, analysis of the electron density map for the hCA I–aprocitentan complex revealed well-defined electron density in the active site, consistent with the structure of the sulfamide ligand (Figure S1). The sulfamide moiety adopted the same binding mode as in hCA II coordinating the zinc ion, and hydrogen bonding was maintained with Thr199, including an additional interaction between one of the sulfamide oxygen atoms and the peptide backbone of Thr199, contributing to the stabilization of the adduct. However, a key difference was noted. In hCA I, the residue corresponding to Thr200 in hCA II is replaced by His200, which resulted in the loss of the hydrogen bond typically formed between the second nitrogen atom of the sulfamide and Thr200 in hCA II (Figure ). The absence of this interaction could contribute to the reduced inhibitory potency of aprocitentan toward hCA I. The bulky aromatic ring on the pyrimidine core is again positioned in a hydrophobic pocket, similar to its placement in hCA II, and this time is stabilized by a perpendicular π-stacking interaction with His200, which may partially compensate for the absence of the sulfamide–Thr200 hydrogen bond. Finally, the tail of the compound did not exhibit significant interactions with the protein, which may further explain the lower inhibitory activity compared to hCA II (Figure ).

3.

X-ray crystal structure of hCA I with bound aprocitentan (PDB ID 9RV0). Residues involved in binding of the inhibitor are also shown. The gray sphere represents the zinc ion in the active site of the protein. Purple dotted lines show the coordination of the zinc ion; van der Waals interactions are shown in blue, hydrogen bonds are shown in red, and π-stacking interactions are shown in green.

In conclusion, the kinetic profiling of aprocitentan across all catalytically active human CA isoforms revealed selective inhibitory activity against cytosolic hCA VII and membrane-associated hCA IX. Although aprocitentan was primarily developed as a dual endothelin receptor antagonist, the presence of a sulfamide moiety in its structure indicates a potential secondary mechanism involving CA inhibition. This dual mechanism of action may contribute synergistically to its antihypertensive effects, offering a broader therapeutic impact. The findings support the hypothesis that CA inhibition may enhance or complement the drug’s primary vasoregulatory function. These results underscore the importance of further mechanistic and clinical investigations to fully elucidate the role of CA modulation in the pharmacological profile of aprocitentan and its potential benefits in the treatment of resistant hypertension.

Glossary

Abbreviations

FDA

Food and Drug Administration

EMA

European Medicines Agency

ET_A

endothelin receptor type A

ET_B

endothelin receptor type B

CA

carbonic anhydrase

CAI

carbonic anhydrase inhibitor

AAZ

acetazolamide

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.5c00421.

• Experimental sections, supplementary figure and X-ray data collection (PDF)

All of the authors approved the final version of the manuscript.

The authors declare no competing financial interest.