The third generation of histamine H2 receptor antagonists such as cimetidine, ranitidine and famotidine are widely used to treat duodenal and gastric ulcers, gastroesophageal reflux disease and pathological hypersecretory disorders such as Zollinger-Ellison syndrome.
Famotidine incorporates a sulfonamide moiety, a bioisostere of the sulfonamide group, a zinc-binding group present in carbonic anhydrase (AC, EC 4.2.1.1) inhibitors (IAC). However, the effects of famotidine on these zinc metalloenzymes have been little investigated. ACs are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and protons.
In humans there are 15 different α-isoforms, with very different subcellular location and tissue distribution.
There are several cytosolic isoforms (hAC I−III, VII, XIII), four membrane-bound isoenzymes (AC IV, IX, VI, apart from three catalytic ones (AC VIII, X and XI).
These enzymes have essential physiological/pathological functions in acid-base homeostasis, electrolyte secretion and ion transport, as they supply bicarbonate and regulate pH in the production of gastric acid within the parietal cells of the stomach, as well as in the production of bile, pancreatic juice, intestinal transport of ions, protection of the digestive tract from extreme pH conditions (too acidic or too basic).
In this special context, the authors investigated whether famotidine shows AC inhibitory effects. All catalytically active isoforms of AC from humans (hAC I−XIV) were included in the study, as well as the two ACs present in the bacterium Helicobacter pylori (α-HpAC, β-HpAC), the etiological agent responsible for the gastric ulcers. They also report on the structure of famotidine determined by X-rays in complex with hCA I and hCA II, which makes it possible to decipher interesting aspects related to its mechanism of inhibition of AC.
The inhibition profile of cytosolic isoforms was very different with famotidine. The isoform most sensitive to inhibition with this drug was hAC VII, with an inhibition constant K i of 3.0 nM. However, the two most widespread cytosolic isoforms hAC I and hAC II showed very different inhibition with this compound: hAC II was effectively inhibited (K i 57.9 nM), while hAC I was more than ten times less sensitive to the inhibition with famotidine, in relation to hAC II, with a K i of 922.4 nM.
Famotidine was a moderate inhibitor of the hAC XIII isoform (K i 171.5 mM) and lost all its activity against hAC III, with a K i > 10 μM. The two mitochondrial isoforms (hAC VA and VB) were poorly inhibited by this sulfamide drug in the micromolar range (K i s of 1.4 and 5.3 μM, respectively).
The membrane-bound isoforms, like the aforementioned cytosolic ones, showed a heterogeneous inhibition pattern. hAC XII, a tumor-associated isoform recently validated as an antitumor target, was effectively inhibited by famotidine with a K i of 45.3 nM.
The second enzyme overexpressed in hypoxic hAC IX tumors was, however, less inhibited by famotidine, with a K i of 126.3 nM. The last two hAC isoforms IV and XIV associated with the membrane, on the other hand, were weakly inhibited by this drug in the high nanomolar range (K i 938.8 and 677.2 nM).
H. pylori , a gram-negative bacteria that thrives at a relatively neutral pH, has been shown to be the cause of chronic gastritis, peptic ulcers, and, more recently, gastric cancer. This gastric bacterium encodes two classes of AC, one α- and the other β. Both enzymes are essential for their survival in the acidic environment of the stomach and recent studies showed that sulfonamides block the growth of the germ in vitro and in vivo.
Thus, in case of interference with these enzymes, famotidine could be used as a new pharmacological tool to treat resistant H. pylori . For this reason, inhibition studies were carried out on the two bacterial ACs. The α class enzyme was effectively inhibited by this compound, with an inhibition constant of 20.7 nM, comparable to acetazolamide (AAZ) used in the clinic.
However, hpβCA was less effectively inhibited by famotidine (K i of 49.8 nM), but also for this enzyme the activity was comparable to AAZ. Famotidine was more active against H. pylori enzymes compared to the two dominant human cytosolic isoforms (hAC I and hAC II), while AAZ was an effective inhibitor for both.
Considering the abundant localization of cytosolic hAC I and II in the digestive tract, as well as their essential functions in pH regulation, high-resolution X-ray crystal structures of famotidine adducts with these enzymes were obtained, in order to know in detail the binding modes of the drug.
Inspection of the initial Fo−Fc electron density maps of both active zones showed well-defined electron density, fully compatible with the presence of famotidine, and surprisingly revealed two possible conformations of the drug for both adducts.
Famotidine coordinates the catalytic zinc ion of the two AC isoforms via a nitrogen atom of the sulfonamide group, displacing the water molecule bound to the zinc/hydroxide ion, which produces a terahedral metal ion coordination geometry, as does other sulfonamide compounds.
The hAC I/famotidine complex showed two different orientations of the inhibitor. The first, presented in green, showed different hydrogen chains that stabilized the enzyme-inhibitor adduct, such as those affecting the guanidine tail and Asn69 or the nitrogen atom near the sulfonamide fraction and Thr199 and His200. However, hydrophobic interactions were almost nonexistent in this complex.
Only one such interaction was found between Leu198 and the methylene chain of famotidine. The opposite orientation of the second structure (in purple), surprisingly, did not show any interaction with the protein side chains.
A totally different situation was observed when analyzing the hCA II active zone. Two possible orientations of famotidine were again observed, both located in the hydrophobic region delimited by a small pocket aligned by the side chains of Phe131, Leu198, and Pro201. The thiazole ring of one orientation of famotidine formed strong hydrophobic interactions with these residues and a water bridge with Pro201 via the nitrogen atom of azomethine.
This portion of the molecule was oriented differently in two conformations: in one, the nitrogen atom participates in several hydrogen chains with Thr199 and Thr200. In the second, an opposite orientation of the nitrogen was observed, without interactions with the protein. Finally, the sulfonamide oxygen participates in a branched oxygen chain with Thr200. Electron density was absent for the guanidine tail of the conformation and was not presented in the complex model.
The structural superposition between the hAC I and hAC II complexes shows that, while the sulfa moieties of famotidine are quite superimposable, the two aliphatic tails present different orientations and, consequently, diverse interactions with the amino acid side chains of the two zones active. Phe131 in hCA II was found to be essential for placing famotidine within the lipophilic side of the active site cavity, which is strongly related to the inhibitory potency of this drug against hCA II.
However, the presence of Leu131 in hCA I led to a loose van der Waals force interaction, which did not force famotidine toward the lipophilic side of the active zone, thus leading to two opposite orientations of the structure and few hydrophobic interactions. with the active zone. These characteristics reflect the loss of inhibitory potency against this isoform compared to hCA II.
In summary , the inhibition pattern of famotidine against all active human ACs was analyzed. The drug shows special efficacy for the cytosolic isoforms hAC II and hAC VII. Furthermore, bacterial enzymes of two classes of H. pylori (hp α AC and hp β AC) were also highly inhibited by this drug, thus raising the possibility that the effective antiulcer effects of famotidine may be due not only to its antagonistic action. of the H2 receptor, but also because, by inhibiting bacterial AC, the survival of the germ within the gastric niche is compromised, as previously demonstrated for acetazolamide. This compound was used as an antiulcer agent with some success, but its side effects due to the inhibition of AC in other tissues ruled out the spread of its clinical use. Famotidine is a much more isoform-selective IAC than acetazolamide and also effectively inhibits bacterial AC. Thus, the authors of this article propose it as a new example for the creation of anti-infective agents with a multifactorial mechanism of action. |
