Carbonic anhydrase inhibitors as emerging drugs for the treatment of obesity

Abstract

Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes in mammals, being involved in numerous biosynthetic processes, including gluconeogenesis, lipogenesis and ureagenesis. It has recently emerged that CA inhibitors (CAIs) targeting the mitochondrial isoforms CA VA and VB have potential as novel antiobesity drugs. This Editorial discusses the biochemical and biological rationale for the use of CAIs in the management of obesity.

Keywords::

• carbonic anhydrase
• mitochondrial enzyme
• obesity
• topiramate

1. Background

Obesity is one of the most common human diseases. However, there are few nontoxic pharmacological approaches for its treatment or prevention [1-4]. Most drugs used for the management of obesity have serious cardiovascular or CNS side effects, which dramatically limit their usefulness [1-4]. Recently, inhibition of carbonic anhydrases (CAs, EC 4.2.1.1) has been proposed as a new antiobesity strategy [4]. Indeed, at least 15 different α-CA isoforms were so far described in vertebrates, where these zinc enzymes play crucial physiological roles [4-10]. Five of them are cytosolic (CA I, CA II, CA III, CA VII and CA XIII), five others are membrane-associated (CA IV, CA IX, CA XII, CA XIV and CA XV) and CA VA and CA VB are mitochondrial and CA VI is secreted in saliva [5-7]. Three acatalytic forms are also known, which were denominated CA-related proteins (CARP), CARP VIII, CARP X and CARP XI, as they lack one or two zinc ligands, which are crucial for the binding of the metal ion [5,6]. Several important physiologic functions are played by the CA isozymes present in organisms all over the phylogenetic tree, related to respiration and transport of CO₂/bicarbonate between metabolizing tissues and the lungs, pH and CO₂ homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions, such as the lipogenesis, gluconeogenesis and ureagenesis among others (in animals), CO₂ fixation (in plants and algae), virulence (in pathogenic bacteria/fungi) and so on [5-7]. The presence of these ubiquitous enzymes in so many tissues and in so different forms represents an attractive goal for the design of inhibitors or activators with biomedical applications [4-9]. Indeed, in addition to the use of the CA inhibitors (CAIs) as antiglaucoma agents, diuretics and anticonvulsants [5], CA IX and XII, isoforms predominantly found in cancerous tissues, were recently validated as antitumor targets, with several monoclonal antibodies and small-molecule inhibitors in advanced clinical evaluations both for the imaging and for treatment of hypoxic solid tumors [6].

Among the α-CA isoforms found in animals, two CA isozymes, CA VA and VB, are present in mitochondria [10,11]. These enzymes are involved in several biosynthetic processes, such as ureagenesis, gluconeogenesis and lipogenesis, both in vertebrates (rodents) and in invertebrates (locust) [10-15]. The provision of enough of the substrate, bicarbonate, in several biosynthetic processes involving pyruvate carboxylase (PC), acetyl CoA carboxylase (ACC) and carbamoyl phosphate synthetases I and II is assured by the catalysis of CO₂ hydration to bicarbonate and protons, involving these mitochondrial isozymes, CA VA and CA VB, probably assisted by the high-activity cytosolic isozyme CA II [4,5,10-17]. Mitochondrial pyruvate carboxylase (PC) is needed for the efflux of acetyl groups from the mitochondria to the cytosol where the fatty acid biosynthesis takes place [16]. Pyruvate is carboxylated to oxaloacetate in the presence of bicarbonate and PC. The bicarbonate needed for this process is generated by CA VA and/or CA VB. The mitochondrial membrane is impermeant to acetyl-CoA, which condenses with oxaloacetate to form citrate, which is thereafter translocated to the cytoplasm with the assistance of the tricarboxylic acid transporter. In the cytosol, citrate is cleaved and regenerates acetyl-CoA and oxaloacetate [4]. As oxaloacetate also cannot cross the mitochondrial membrane, its decarboxylation regenerates pyruvate, which can be then transported into the mitochondria by means of the pyruvate transporter [4,5,16,17]. The acetyl-CoA thus generated in the cytosol is in fact used for the de novo lipogenesis, by carboxylation in the presence of ACC and bicarbonate, with the formation of malonyl-CoA. The bicarbonate needed in this process is furnished by the CA II – catalyzed conversion of CO₂ to bicarbonate. Subsequent biosynthetic steps involving the sequential transfer of acetyl groups lead to longer chain fatty acids [4,15-17]. As a whole, three CA isoforms are critical to the entire process of fatty acid biosynthesis: CA VA/VB within the mitochondria (to provide enough substrate to PC) and CA II within the cytosol (for providing sufficient substrate to ACC) [4]. It was demonstrated that inhibition of such CAs by sulfonamides, the main class of CAIs in clinical use [18], decrease lipogenesis in adipocytes in cell culture [12-14]. In such earlier experiments [10-15], an indiscriminate inhibition of all CA isozymes present in these tissues was achieved by using inhibitors with no selectivity for the mitochondrial enzymes, but they nonetheless demonstrated an effect for the inhibition of lipogenesis [12-14]. As it will be shown shortly, in the last decade many CA VA/VB selective inhibitors have been reported, which may lead to compounds with a more effective antiobesity profile [19-28].

2. CA inhibitors as antiobesity agents

Several studies have provided evidence that CAIs of the sulfonamide/sulfamate type have potential as antiobesity drugs [4,5,29-34]. Topiramate is an antiepileptic drug possessing potent anticonvulsant effects due to a multifactorial mechanism of action: blockade of sodium channels and kainate/AMPA receptors, CO₂ retention secondary to inhibition of the red cell and brain CA isozymes as well as enhancement of GABA-ergic transmission [4]. A side effect of this drug observed in obese patients was the loss of body weight, although no pharmacological explanation of this phenomenon has been provided [4]. Furthermore, topiramate was shown to reduce energy and fat gain in lean (Fa/?) and obese (fa/fa) Zucker rats [29]. It was demonstrated thereafter that topiramate is a very potent inhibitor of several CA isozymes, such as CA II, VA, VB, VI, VII, XII and XIII [5,8,9], and the X-ray crystal structure of its complex with CA II and CA I has been determined [8,35], revealing the molecular interactions that explain the high affinity of this compound for the enzyme active site. Topiramate acts as a highly effective inhibitor of the mitochondrial isozymes CA VA and CA VB involved in lipogenesis, as shown above, which provides a rationale for the use of this compound to control weight loss and obesity, and also explains the side effects observed in the obese epileptic patients treated with it [4,5]. Indeed, Qnexa, a combination of topiramate and low dose of phentermine, entered clinical trials for the treatment of obesity [32,33]. In a 56-week Phase III such trial, overweight or obese adults (aged 18 – 70 years), with a body mass index of 27 – 45 kg/m² and two or more comorbidities (hypertension, dyslipidemia, diabetes or prediabetes, as well as abdominal obesity) were randomly assigned to placebo, or once-daily Qnexa (phentermine 7.5 mg plus topiramate 46.0 mg), or once-daily Qnexa double dose (phentermine 15.0 mg plus topiramate 92.0 mg) in a 2:1:2 ratio, in 93 centers in the US. The drugs were administered orally [33]. Primary endpoints were the percentage change in bodyweight and the proportion of patients achieving at least 5% weight loss. A significant number of the treated patients achieved a weight loss of at least 5%, and it was concluded that Qnexa might be a valuable treatment for obesity [33,34]. More precisely, on the high-dose Qnexa, the weight loss was of 14.4% and on the low dose of 6.7% of the baseline body weight [34].

Zonisamide is another antiepileptic drug used as adjunctive therapy for refractory partial seizures [5]. As topiramate, it has multiple mechanisms of action and exhibits a broad spectrum of anticonvulsant activity [5]. Similar to topiramate, recent clinical studies have demonstrated additional potential for therapeutic use in neuropathic pain, bipolar disorder, migraine, Parkinson's disease as well as obesity and eating disorders [30]. Zonisamide is an aliphatic sulfonamide, which also potently inhibits cytosolic and mitochondrial CAs involved in lipogenesis [9]. Furthermore, zonisamide in conjunction with a reduced-calorie diet (deficit of 500 kcal/day) resulted in an additional mean 5-kg (6 – 10% of the initial body weight) weight loss compared with diet alone in obese female patients [30]. However, due to their potent inhibitory effects against most CA isozymes, topiramate and zonisamide may lead to a range of side effects when used as monotherapy [5]. The most common of them are formation of kidney stones, metabolic acidosis and enhanced diuresis [4-6]. It is thus essential to develop CAIs with high selectivity for the mitochondrial enzymes.

3. Future developments and conclusions

The antiobesity effects of topiramate and zonisamide were discovered serendipitously, but they could be rationalized considering the potent inhibitory effects of the two compounds against the mitochondrial CA isoforms involved in lipogenesis, CA VA and CA VB [4,5]. Recently, a rational drug design approach of antiobesity CAIs has been reported, by examining the role of mitochondrial CAs in the metabolism of pyruvate, acetate and succinate [28]. An electrochemical approach of wired mitochondria to analytically measure metabolic energy conversion has been developed, which led to the observation that diverse CAIs of the sulfonamide type have very different metabolic profiles (all the investigated compounds were low nanomolar CA VA/VB inhibitors) [28]. This study showed pyruvate metabolism to be the most affected pathway by the sulfonamide CAIs, followed by the fatty acid metabolism, whereas the succinate metabolism was least affected [28]. This new approach [28] allows for a relatively easy and inexpensive way of detecting effective antiobesity compounds among the CA VA/VB inhibitors described so far in the literature [4,5,18-25] avoiding the highly expensive and time-consuming animal experiments.

Another highly important finding supporting the usefulness of CAIs in the management of obesity and related comorbidities was recently reported [36]. In diabetes patients, who also frequently suffer from obesity, an increased mitochondrial oxidative stress was shown to be implicated as a mechanism for hyperglycemia-induced pericyte (PC) loss, which is the prerequisite leading to blood–brain barrier disruption [36]. The same authors proved that mitochondrial CAs regulate the oxidative metabolism of glucose and thus play an important role in the generation of reactive oxygen species and oxidative stress. Their inhibition with topiramate thus leads to a rescue cerebral PC loss caused by diabetes-induced oxidative stress, preserving blood–brain barrier integrity [36]. Thus, in addition to having a significant antiobesity effect by inhibiting lipogenesis, topiramate (and presumably all the potent CA VA/VB inhibitors) reduces diabetes-induced oxidative stress in the brain and rescues cerebral blood–brain barrier damage, ensuring a doubly beneficial effect in obesity patients.

All data presented here strongly suggest that inhibition of mitochondrial enzymes CA VA and CA VB (probably in conjunction with that of the ubiquitous cytosolic isoform CA II) [37] represents a valid strategy for obtaining antiobesity drugs with a novel mechanism of action, which reduces lipogenesis and mitochondrial oxidative stress associated with many obesity comorbidities. Although no new drugs specifically acting on these pathways were yet developed (with clinical benefits observed so far only for some antiepileptics, such as topiramate and zonisamide), recent biochemical and biological findings allow for some optimisms in this scarcely investigated field of CA research.

Declaration of interest

The author declares no conflict of interest. No financial sponsorship or research grants regarding antiobesity CA inhibitors were received.