International Journal of

Molecular Sciences Review

Non-Classical Inhibition of Carbonic Anhydrase Carrie L. Lomelino 1 , Claudiu T. Supuran 2 and Robert McKenna 1, * 1 2

*

Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32611, USA; [email protected] Neurofarba Department, University of Florence, Piazza di San Marco, Firenze 50019, Italy; [email protected] Correspondence: [email protected]; Tel.: +1-352-294-8395

Academic Editor: Ester Boix Received: 31 May 2016; Accepted: 7 July 2016; Published: 16 July 2016

Abstract: Specific isoforms from the carbonic anhydrase (CA) family of zinc metalloenzymes have been associated with a variety of diseases. Isoform-specific carbonic anhydrase inhibitors (CAIs) are therefore a major focus of attention for specific disease treatments. Classical CAIs, primarily sulfonamide-based compounds and their bioisosteres, are examined as antiglaucoma, antiepileptic, antiobesity, antineuropathic pain and anticancer compounds. However, many sulfonamide compounds inhibit all CA isoforms nonspecifically, diluting drug effectiveness and causing undesired side effects due to off-target inhibition. In addition, a small but significant percentage of the general population cannot be treated with sulfonamide-based compounds due to a sulfa allergy. Therefore, CAIs must be developed that are not only isoform specific, but also non-classical, i.e. not based on sulfonamides, sulfamates, or sulfamides. This review covers the classes of non-classical CAIs and the recent advances in the development of isoform-specific inhibitors based on phenols, polyamines, coumarins and their derivatives. Keywords: carbonic anhydrase; carboxylate; coumarin; non-classical

1. Introduction Carbonic anhydrase (CA, EC 4.2.1.1) is a family of zinc metalloenzymes that catalyze the reversible interconversion of carbon dioxide and water to a bicarbonate and a proton. As such, CA activity is important for several physiological processes such as pH regulation, CO2 homeostasis, respiration, bone resorption, and tumorigenesis [1,2]. The CA family is subdivided into six classes based upon their structural fold, which also often correlate to their predominant organisms of expression. These classes include αCA expressed in vertebrates and algae; βCA in plants and prokaryotes; γCA in archaea; δCA and ζCA expressed in marine diatoms; and η in protozoa. In humans, there are 16 isoforms of αCA expressed that vary by localization and catalytic activity: CA I, CA II, CA III, CA VII, CA XIII are cytosolic; CA IV, CA IX, CA XII, CA XIV, CA XV membrane-bound; CA Va and CA Vb mitochondrial; and CAVI secreted in saliva and colostrum [3–5]. In addition, there are three catalytically inactive forms (CA VIII, CA X, and CA XI) referred to as CA-related proteins (CARPs) [5]. The αCA active site structure is conserved and is conically shaped with a zinc atom located at the base, which is coordinated by three histidine residues (His94, His96, His119) and a water/hydroxide ion. The catalytic mechanism of CA occurs in two steps. In the hydration direction, first there is a nucleophilic attack of carbon dioxide by the zinc-bound hydroxide ion, resulting in a zinc-bound bicarbonate that is subsequently displaced by a water molecule [4,6,7]. The enzyme active site is then regenerated to a zinc-bound hydroxide ion by a proton transfer mechanism from the zinc-bound water molecule to bulk solvent, facilitated by an ordered water wire within the active site and His64 acting as a proton donor/acceptor shuttle residue [8–11]. Int. J. Mol. Sci. 2016, 17, 1150; doi:10.3390/ijms17071150

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1.1. CA Inhibition Various isoforms of CA have been identified as therapeutic targets for several diseases, see Table 1. Therefore, the design of isoform-specific inhibitors is studied for the development of new and Int. J. Mol. Sci. 2016, 17, 1150 2 of 12 improved treatments [6,12–23]. 1.1. CA Inhibition

Table 1. Carbonic anhydrase (CA) related diseases with associated isoform targets.

Various isoforms of CA have been identified as therapeutic targets for several diseases, see Table 1. Therefore, the design of isoform-specific inhibitors is studied for the development of new Disease CA Isoform Target and improved treatments [6,12–23].

Glaucoma

CA II, CA IV, CA XII

Table 1. Carbonic anhydrase (CA) related diseases with Cancer CA IX,associated CA XII isoform targets.

EpilepsyDisease Antineuropathic pain Glaucoma Obesity Cancer Epilepsy Antineuropathic pain inhibited byObesity compounds

CA VIITarget CA Isoform VIICA XII CA ΙΙ, CA CA IV, VA XII CACA IX, CA CA VII CA VII a sulfonamide-based CA VA

CA is classically with (SO2 NH2 ) zinc-binding group (ZBG) or their bioisosteres (sulfamates and sulfamides). Sulfonamides bind in a tetrahedral CA is classically inhibited by catalytic compounds a sulfonamide-based (SO2NH 2) zinc-binding geometry, interacting directly with the zincwith in their deprotonated form, and inhibit CA activity group (ZBG) or their bioisosteres (sulfamates and sulfamides). Sulfonamides bind in a tetrahedral by displacing the zinc-bound water/hydroxide ion. There are currently several sulfonamide-based CA geometry, interacting directly with the catalytic zinc in their deprotonated form, and inhibit CA inhibitors clinically available for the treatment of glaucoma, edema, epilepsy, and altitude sickness [4]. activity by displacing the zinc-bound water/hydroxide ion. There are currently several However, the design isoform-specific inhibitors istreatment complicated by theedema, structural homology sulfonamide-based CAof inhibitors clinically available for the of glaucoma, epilepsy, betweenand thealtitude 16 CAsickness isoforms, [4].which is particularly high within the active site (Figure 1A). Consequently, the design of isoform-specific is complicatedcausing by the structural homology the availableHowever, CA inhibitors act systemically and inhibitors bind nonspecifically, a range of undesired side between the 16 CA isoforms, which is particularly high within the active site (Figure 1A). effects, such as fatigue and nausea, due to off-target inhibition. As CA II is the most physiologically Consequently, the available CA inhibitors act systemically and bind nonspecifically, causing a range abundant isoform, it is often regarded as the predominant off-target isozyme of which inhibition is to of undesired side effects, such as fatigue and nausea, due to off-target inhibition. As CA ΙΙ is the be avoided It has been suggested classical carbonic anhydrase most[3,4,24,25]. physiologically abundant isoform, it isthat oftennonspecific, regarded as the predominant off-target isozymeinhibitors of (CAIs), which such as acetazolamide, be delivered large doses to increase the probability of inhibiting a inhibition is to be avoided [3,4,24,25].in It has been suggested that nonspecific, classical carbonic anhydrase inhibitors (CAIs), such as acetazolamide, be delivered in large doses increase the disease-associated target isoform. However, the inhibitor distribution would betodisproportionate in probability of inhibiting a disease-associated target isoform. However, the inhibitor distribution relation to the levels of isoform expression. As such, CA II would sequester the free inhibitor and be disproportionate in relation to the levels of isoform expression. As such, CA ΙΙ would preventwould binding to the target isoform. Therefore, the development of isoform specific inhibitors will sequester the free inhibitor and prevent binding to the target isoform. Therefore, the development of preventisoform side effects and improve the distribution of CA inhibitors. specific inhibitors will prevent side effects and improve the distribution of CA inhibitors.

Figure 1. (A) Overlay of catalytically active human Carbonic anhydrase (CA) isoforms; (B) Surface

Figure 1. (A) Overlay of catalytically active human Carbonic anhydrase (CA) isoforms; (B) Surface representation of CA ΙΙ with hydrophobic and hydrophilic regions colored orange and purple, representation of CA II with hydrophobic hydrophilic regions orange and purple, respectively. Isoform-unique residues withinand the active site are colored bluecolored and labeled. respectively. Isoform-unique residues within the active site are colored blue and labeled. The design of isoform-selective compounds has been improved by the identification of a selective pocket in human CA active sites and further development of drug design approaches. The The design of isoform-selective compounds has been improved by the identification of a selective CA active site can be divided into two halves characterized by hydrophobic and hydrophilic pocket in humanWhile CA active sites and further development ofzinc drug approaches. The CA active residues. active site residues surrounding the catalytic aredesign primarily conserved between

site can be divided into two halves characterized by hydrophobic and hydrophilic residues. While active site residues surrounding the catalytic zinc are primarily conserved between isoforms, unique

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residues are located 10–15 Å from the zinc, designated as the selective pocket (Figure 1B) [26–28]. Therefore, compounds can be specifically designed to exploit interactions with these residues utilizing the tail method. In this approach, a lead compound with a high-affinity ZBG is identified and the “tail” ofInt. the drug J. Mol. Sci. modified 2016, 17, 1150 in order to promote interactions with isoform-unique residues3 or of 12alter the solubility and physico-chemical properties of the compound. Common variations include the length isoforms, unique residues are located 10–15 Å from the zinc, designated as the selective pocket of the tail and class or characteristics of the chemical substituents used to derivatize the inhibitor [5,29]. (Figure 1B) [26–28]. Therefore, compounds can be specifically designed to exploit interactions with Recently, theresidues successutilizing of the tail method to In produce isoform specific inhibitors highlighted these the tail method. this approach, a lead compound with a was high-affinity ZBG by the design of a CAthe IX-specific tested clinicalinteractions trials for the treatment is SLC-0111, identified and “tail” of inhibitor the drugcurrently modified being in order to in promote with of breastisoform-unique cancer [30]. residues or alter the solubility and physico-chemical properties of the compound. Common variations include the length of the tail and class or characteristics of the chemical substituentsCA used to derivatize the inhibitor [5,29]. Recently, the success of the tail method to 1.2. Non-Classical Inhibition produce isoform specific inhibitors was highlighted by the design of SLC-0111, a CA IX-specific

Although have studied as CAIs inhibitor sulfonamide-based currently being tested incompounds clinical trials for the been treatment of breast cancerfor [30].several decades and several inhibitors are in clinical use, 3%–6% of the general population cannot be treated with such 1.2. Non-Classical CAallergies. Inhibition Sulfonamide treatments can result in either a type 1 immunological compounds due to sulfa reaction, commonly caused by immunoglobulin E (IgE) developed against the inhibitor, Although sulfonamide-based compounds have been antibodies studied as CAIs for several decades and several 1 inhibitors areininwhich clinicalmetabolites use, 3%–6% ofofthe population cannot treated with suchor T cell or a non-type response thegeneral inhibitor interact withbe a native protein compounds due to sulfa Sulfonamide treatments result in either a type 1 to stimulate a response [31]. Theallergies. incidence of adverse reactionscan to sulfonamide-based compounds immunological reaction, commonly caused by immunoglobulin E (IgE) antibodies developed increases with age and is more commonly exhibited in women [32]. Although reactions are most often against the inhibitor, or a non-type 1 response in which metabolites of the inhibitor interact with a seen to sulfonamide have abeen reported allergic responses to non-antibiotic native protein antibiotics, or T cell tocases stimulate response [31].for The incidence of adverse reactions to CAIs, such assulfonamide-based acetazolamide. Side effectsincreases of commonly CAIs coverinawomen wide range compounds with ageused and issulfonamide more commonly exhibited [32]. from Although reactions often seenshock to sulfonamide cases have [33]. been Therefore, reported for there is epidermis rash to nauseaare to most anaphylactic or acute antibiotics, respiratory failure allergic responses non-antibiotic CAIs, such as but acetazolamide. Side effects commonly a need for CAIs that aretonot only isoform-specific, also non-classical so of they are notused based on a sulfonamide CAIs cover a wide range from epidermis rash to nausea to anaphylactic shock or acute sulfonamide, sulfamate, or sulfamide ZBG. respiratory failure [33]. Therefore, there is a need for CAIs that are not only isoform-specific, but also Several classes of compounds have recently been identified as non-classical CAIs, including non-classical so they are not based on a sulfonamide, sulfamate, or sulfamide ZBG. phenols, polyamines, carboxylic acids, coumarins andidentified their derivatives, in addition to fullerenes Several classes of compounds have recently been as non-classical CAIs, including (Figure phenols, 2). While it is possible for acids, non-classical to maintain traditional tetrahedral polyamines, carboxylic coumarinsinhibitors and their derivatives, in the addition to fullerenes (Figure 2). Whiledirectly it is possible non-classical to maintain traditional geometry by binding to thefor catalytic zinc,inhibitors the classes covered the in this reviewtetrahedral also participate geometry by binding directly toFor the catalytic zinc, the classes covered this been reviewidentified also participate in different binding mechanisms. example, non-classical CAIs in have that inhibit in different binding mechanisms. For example, non-classical CAIs have been identified that inhibit CA catalytic activity by anchoring to the zinc-bound water/hydroxide ion or binding to the enzyme CA catalytic activity by anchoring to the zinc-bound water/hydroxide ion or binding to the enzyme outside outside the active site, occluding the entry of substrate and preventing catalysis. the active site, occluding the entry of substrate and preventing catalysis.

Figure 2. (A) Phenol; (B) Polyamine: n is an integer, R is either an N or C atom; (C) Carboxylic acid

Figure (D) 2. Coumarin (A) Phenol; (B) Polyamine: n is an integer, R is either an N or C atom; and hydrolysis product; (E) Sulfocoumarin and hydrolysis product; (F) Fullerene (C) Carboxylic acid; (D) Coumarin and hydrolysis product; (E) Sulfocoumarin and hydrolysis product; derivative. (F) Fullerene derivative.

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2. Non-Classical Inhibitor Classes 2.1. Phenols

Phenol is a CAI that acts as a competitive substrate with a micromolar affinity for CA in the 2.1. Phenols forward hydration reaction [34]. The OH moiety anchors to the zinc-bound water/hydroxide ion Phenol is a CAI that acts as a competitive substrate with a micromolar affinity for CA in the through a hydrogen bond while the phenyl functional group participates in van der Waals interactions forward hydration reaction [34]. The OH moiety anchors to the zinc-bound water/hydroxide ion with the hydrophobic half bond of thewhile activethe site,phenyl preventing the binding carbon dioxide 3) [34,35]. through a hydrogen functional group of participates in van(Figure der Waals Alternative to sulfonamide-based compounds, phenolic compounds have been shown to inhibit interactions with the hydrophobic half of the active site, preventing the binding of carbon dioxide CA independent the protonation Phenol inhibits all 13 catalytically active humanhave isoforms (Figure 3)of[34,35]. Alternativestate. to sulfonamide-based compounds, phenolic compounds been with shown to inhibit CA independent of the protonation state. Phenoland inhibits 13 catalytically varying affinities, so phenol can be employed as a lead compound new all derivatives can beactive designed human isoforms with varying affinities, phenolitself can be as a lead structure, compound the and inclusion new that improve isoform specificity [36]. As so phenol is employed a rather compact derivatives can be designed that improve isoform specificity [36]. As phenol itself is a rather of additional rings or other functional groups that lengthen the compound are expected to increase compact structure, the inclusion of additional rings or other functional groups that lengthen the selectivity by promoting interactions with residues of the selective pocket. For example, the addition compound are expected to increase selectivity by promoting interactions with residues of the of a single chemical substituent, such as a carboxy moiety, has been shown to increase CA inhibition selective pocket. For example, the addition of a single chemical substituent, such as a carboxy several fold with binding affinities in the low- to sub-micromolar range [37]. Phenolic ester inhibitors moiety, has been shown to increase CA inhibition several fold with binding affinities in the low- to have sub-micromolar been designedrange to increase the length the compounds, exhibiting binding profiles [37]. Phenolic esterof inhibitors have been designed to selective increase the length of the of sub-micromolar thatselective are simultaneously poor inhibitors of off-targetaffinities CA II [38].that are compounds, affinities exhibiting binding profiles of sub-micromolar Polyphenols structurally complex natural products are similarly shown to exhibit simultaneouslyand poormore inhibitors of off-target CA ΙΙ [38]. and more structurally complex natural productsphenol are similarly shown to exhibit improvedPolyphenols binding affinities in comparison to the lead compound, [37]. Phenol-based natural improved binding affinities in comparison to the lead compound, phenol [37]. Phenol-based natural product discovery provides a promising direction for CA inhibition since these compounds are derived discovery provides a promising for CA inhibitionboth sincea these compounds are fromproduct plants and are already ingested in thedirection human diet, providing nontoxic and sulfur-free derived from plants and are already ingested in the human diet, providing both a nontoxic and compound. Several of these phenol-based natural products show specificity for CA VII and are sulfur-free compound. Several of these phenol-based natural products show specificity for CA VII recognized to also play a role in antioxidant activity, which in combination with CA inhibitory activity and are recognized to also play a role in antioxidant activity, which in combination with CA couldinhibitory develop activity into novel treatments for neurodegenerative diseases [39]. could develop into novel treatments for neurodegenerative diseases [39].

Figure 3. Phenol-based compounds benzene-1,4-diol (cyan) and resorcinol (magenta) in complex

Figure 3. Phenol-based compounds benzene-1,4-diol (cyan) and resorcinol (magenta) in complex with with CA ΙΙ (gray) (PDBs: 4E3H and 4E49, respectively) [40]. CA II (gray) (PDBs: 4E3H and 4E49, respectively) [40].

2.2. Polyamines

2.2. Polyamines Polyamines are polycationic, aliphatic molecules that were originally expected to act as activators of are CA polycationic, due to the activating properties observed for amino acidsexpected and amines. However, Polyamines aliphatic molecules that were originally to act as activators activity assays determined several polyamines, such as spermine, spermidine, and their derivatives, of CA due to the activating properties observed for amino acids and amines. However, activity assays exhibit inhibitory properties of CA. The crystal structure of spermine in complex with CA ΙΙ determined several polyamines, such as spermine, spermidine, and their derivatives, exhibit inhibitory elucidated the binding mechanism for polyamines; similar to that of phenolic compounds, a properties of CA. The crystal structure of spermine in complex with CA II elucidated the binding terminal ammonium group anchors to the zinc-bound water/hydroxide ion through a hydrogen mechanism for polyamines; to thatbecause of phenolic compounds, a terminal group bond. Polyamine binding similar is also unique the compound binding relies onammonium a network of anchors to the zinc-bound water/hydroxide ion through a hydrogen bond. Polyamine binding is also hydrogen bonding, with key interactions occurring between the inhibitor and Thr199, a residue unique because the compound binding relies on a network of hydrogen bonding, with key interactions occurring between the inhibitor and Thr199, a residue conserved between all isoforms, and the other

conserved between all isoforms, and the other terminal amine with active site residues. Polyamines, such as spermine in adduct with hCA ΙΙ, are observed to coil within the active site with the aliphatic portion stabilized by van der Waals interactions (Figure 4) [41]. Variations in the length of the aliphatic chain and the number of amine substituents are seen to Int. J. Mol. Sci. 2016, 17, 1150 5 of 13 affect CA binding affinities between the different isoforms, indicating the potential of polyamine compounds to be developed into selective CAIs. In addition, the polycationic nature of polyamines makes them unable to pass cell membranes, so these inhibitors can be used to target CA isoforms terminal amine with active site residues. Polyamines, such as spermine in adduct with hCA II, are with extracellular catalytic domains. Therefore, polyamines show therapeutic potential in relation to observed to coil within the active site with the aliphatic portion stabilized by van der Waals interactions the inhibition of cancer-associated CA IX and CA XII, which is highlighted by the selectivity of Int. J. 4) Mol. Sci. 2016, 17, 1150 5 of 12 (Figure [41]. natural product polyamine fragments over cytosolic off-target isoforms [42]. conserved between all isoforms, and the other terminal amine with active site residues. Polyamines, such as spermine in adduct with hCA ΙΙ, are observed to coil within the active site with the aliphatic portion stabilized by van der Waals interactions (Figure 4) [41]. Variations in the length of the aliphatic chain and the number of amine substituents are seen to affect CA binding affinities between the different isoforms, indicating the potential of polyamine compounds to be developed into selective CAIs. In addition, the polycationic nature of polyamines makes them unable to pass cell membranes, so these inhibitors can be used to target CA isoforms with extracellular catalytic domains. Therefore, polyamines show therapeutic potential in relation to the inhibition of cancer-associated CA IX and CA XII, which is highlighted by the selectivity of natural product polyamine fragments over cytosolic off-target isoforms [42].

Figure 4. Spermine (salmon) in complex with CA ΙΙ (gray) (PDB:3KWA) [41].

Figure 4. Spermine (salmon) in complex with CA II (gray) (PDB:3KWA) [41].

2.3. Carboxylic Acids

Variations in the length of the aliphatic chain and the number of amine substituents are seen Carboxylic acids represent a class of compounds that inhibit metalloenzymes through various to affect CA binding affinities between the different isoforms, indicating the potential of polyamine mechanisms of action, such as coordinating to the metal ion in a mono- or bidentate manner. This is compounds to be into selective In addition, the polycationic nature of[43]. polyamines also the case fordeveloped CAs, for which a numberCAIs. of inhibition mechanisms have been observed First, makes them unable to pass cell membranes, so these inhibitors can be used to target CA isoforms with carboxylate compounds can directly bind to the catalytic zinc and displace the bound extracellular catalytic domains. Therefore, polyamines show therapeutic potential in relation to the water/hydroxide ion, as seen in classical sulfonamide inhibition (Figure 5). inhibition of cancer-associated CA IX and CA XII, which is highlighted by the selectivity of natural product polyamine fragments over cytosolic off-target isoforms [42]. Figure 4. Spermine (salmon) in complex with CA ΙΙ (gray) (PDB:3KWA) [41].

2.3. Carboxylic Acids 2.3. Carboxylic Acids

Carboxylic acids represent a class of compounds that inhibit metalloenzymes through various Carboxylic acidssuch represent a class of compounds that inhibit metalloenzymes through various mechanisms of action, as coordinating to the metal ion in a monoor bidentate manner. This is mechanisms of action, such as coordinating to the metal ion in a mono- or bidentate manner. This is also the case for CAs, for which a number of inhibition mechanisms have been observed [43]. First, also the case for CAs, for which a number of inhibition mechanisms have been observed [43]. First, carboxylate compounds can directly bind to the catalytic zinc and displace the bound water/hydroxide carboxylate compounds can directly bind to the catalytic zinc and displace the bound ion, as seen in classical sulfonamide inhibition (Figure 5). water/hydroxide ion, as seen in classical sulfonamide inhibition (Figure 5).

Figure 5. (A) CA ΙΙ (gray) in complex with cholic acid (yellow) (PDB:4PXX); (B) Surface representation of CA II in complex with cholic acid (yellow).

Secondly, some carboxylates anchor to the zinc-bound water/hydroxide ion through a hydrogen bond, similar to the binding mechanism seen in phenol-based compounds (Figure 6). However, these inhibitors are predicted to bind primarily in their anionic state [40].

Figure 5. (A) CA ΙΙ (gray) in complex with cholic acid (yellow) (PDB:4PXX); (B) Surface

Figure 5. (A) CA II (gray) in complex with cholic acid (yellow) (PDB:4PXX); (B) Surface representation representation of CA II in complex with cholic acid (yellow). of CA II in complex with cholic acid (yellow).

Secondly, some carboxylates anchor to the zinc-bound water/hydroxide ion through a hydrogen bond, similar to the binding mechanism seen in phenol-based compounds (Figure 6). However, these inhibitors are predicted to bind primarily in their anionic state [40].

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Secondly, some carboxylates anchor to the zinc-bound water/hydroxide ion through a hydrogen bond, similar to the binding mechanism seen in phenol-based compounds (Figure 6). However, these inhibitors are predicted to bind primarily in their anionic state [40]. Int. J. Mol. Sci. 2016, 17, 1150

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Figure 6. (A) CA ΙΙ (gray) in complex with 2,6-dihydroxybenzoic (green, PDB:4E3F) [40]; (B) CA ΙΙ

Figure 6. (A) CA II (gray) in complex with 2,6-dihydroxybenzoic (green, PDB:4E3F) [40]; (gray) in complex with ligands (E)-3-(4-methoxyphenyl)but-2-enoic acid (magenta) and (B) (E)-3-(4-chlorophenyl)but-2-enoic CA II (gray) in complex with ligands (E)-3-(4-methoxyphenyl)but-2-enoic acid (magenta) and acid (yellow) (PDBs: 5FNM and 5FLS, respectively) [44]; (E)-3-(4-chlorophenyl)but-2-enoic acid (yellow) (PDBs: 5FNM and 5FLS, respectively) [44]; (C) Overlay (C) Overlay of carboxylic acid–based compounds in CA ΙΙ surface. of carboxylic acid–based compounds in CA II surface.

Lastly, one carboxylate derivative was found to bind CA outside the active site, in a pocket adjacent thecarboxylate entrance (Figure 7). This was binding blocks His64,CA the outside proton shuttle residue, in in its a “out” Lastly, to one derivative found to bind the active site, pocket conformation, leading (Figure to the inhibition of catalytic activity [45]. the proton shuttle residue, in its “out” adjacent to the entrance 7). This binding blocks His64, The scaffold of to carboxylic acid–based inhibitors can vary conformation, leading the inhibition of catalytic activity [45].in both size and chemical properties, allowing interactions with either the hydrophobic or hydrophilic half of the active site, in addition to The scaffold of carboxylic acid–based inhibitors can vary in both size and chemical properties, isoform-unique residues of the selective pocket. Furthermore, the orientation of functional groups in allowing interactions with either the hydrophobic or hydrophilic half of the active site, in addition relation to the carboxylic acid ZBG has been shown to be an important factor in binding due to the to isoform-unique residues of the selective pocket. Furthermore, the orientation of functional groups possibility of steric hindrance, further promoting selectivity based upon the size of amino acids in relation to active the carboxylic acid ZBG has been that shown to be an important binding due to lining the site cavity [46]. Compounds extend further from the factor active in site have been the shown possibility of steric further promoting selectivity based upon the fold, size highlighting of amino acids to increase thehindrance, binding affinity of carboxylic acid–based inhibitors over 100 lining the active site cavity [46]. Compounds that extend further from the active site have the importance of interactions with the selective pocket residues. For example, structures ofbeen CA ΙΙshown in to increase the binding affinity of carboxylic acid–based inhibitors over 100 fold, highlighting complex with butenoic acid inhibitors (PDB:5FNM and 5FLS) exhibit Ki values between 700–900 the importance the acid selective pocket residues.only Forhas example, structures of5CA µM, whileofa interactions more compactwith benzoic derivative (PDB:4E3F) a Ki of approximately mM.II in As these compounds all anchor through the water/hydroxide ion and formµM, complex withthree butenoic acid inhibitors (PDB:5FNM and zinc-bound 5FLS) exhibit Ki values between 700–900 hydrogen with conserved Thr199, the additional van ader interactions formed while a more bonds compact benzoic acidresidue derivative (PDB:4E3F) only has KiWaals of approximately 5 mM. As by the butenoic acid derivatives with side chains in the hydrophobic half of the active site dictate this these three compounds all anchor through the zinc-bound water/hydroxide ion and form hydrogen increase in specificity (Figure 6). bonds with conserved residue Thr199, the additional van der Waals interactions formed by the butenoic

acid derivatives with side chains in the hydrophobic half of the active site dictate this increase in specificity (Figure 6).

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Figure 7. The 2-[(S)-benzylsulfinyl] benzoic acid (periwinkle) in complex with CA ΙΙ (gray)

Figure 7. The 2-[(S)-benzylsulfinyl] benzoic acid (periwinkle) in complex with CA II (gray) (PDB:4QY3) [45]. (PDB:4QY3) [45].

Several other scaffolds of carboxylic acid compounds have been discovered that exhibit isoform specificity. For example, containing a cyclic imide scaffold the Several other scaffolds inhibitors of carboxylic acid compounds have been selectively discoveredinhibit that exhibit cancer-associated isoforms CA IX and CA XII over off-target, cytosolic CA Ι and CA ΙΙ. These isoform specificity. For example, inhibitors containing a cyclic imide scaffold selectively inhibit acid compounds exhibit selective inhibition for the transmembrane the carboxylic cancer-associated isoforms CA more IX and CA XII over off-target, cytosolic CACAI isoforms and CA II. thancarboxylic sulfonamide containing same cyclic inhibition imide scaffold [47].transmembrane Inhibitors withCA These acidinhibitors compounds exhibit the more selective for the heteroaryl-pyrazole and hydroxy- oxoindolin-ylidene scaffolds show selectivity for CA Ι over CA ΙΙ. isoforms than sulfonamide inhibitors containing the same cyclic imide scaffold [47]. Inhibitors with CA Ι is studied as a target for the treatment of diabetic macular edema, a condition which left heteroaryl-pyrazole and hydroxy- oxoindolin-ylidene scaffolds show selectivity for CA I over CA II. untreated can lead to loss of eyesight [46]. Lastly, carboxylic acid compounds incorporating a CA I is studied as a target for the treatment of diabetic macular edema, a condition which left phthalic anhydride/phthalimide scaffold show selectivity for CA VII over CA Ι and CA ΙΙ. As CA VII untreated can lead to loss of eyesight [46]. Lastly, carboxylic acid compounds incorporating a phthalic activity has been associated with epileptic seizures, CA VII selective inhibitors present a new anhydride/phthalimide scaffold show selectivity for CA VII over CA I and CA II. As CA VII activity method of treatment [48].

has been associated with epileptic seizures, CA VII selective inhibitors present a new method of treatment [48]. 2.4. Coumarins Coumarins were first discovered through a general screen of natural product CAIs and quickly 2.4. Coumarins

became of interest due to the observance of CA inhibition with micromolar to nanomolar affinity for Coumarins firstdespite discovered through a general screen of natural productfrom CAIs andother quickly all active αCAwere isoforms the lack of a canonical ZBG. Coumarins are unique many became interest dueare to considered the observance of CA micromolar to the nanomolar affinity CAIsof because they prodrugs thatinhibition can only with bind the enzyme in form of their hydrolysis product [49,50]. Coumarins are proposed to first bind in the active site, undergo for all active αCA isoforms despite the lack of a canonical ZBG. Coumarins are unique from many hydrolysis due to the activity of prodrugs CA, and then to prevent steric hindrance. Due to of other CAIs because theyesterase are considered thatreorient can only bind the enzyme in the form this required product chemical [49,50]. transformation, coumarins are considered suicide and undergo their their hydrolysis Coumarins are proposed to first bind in compounds the active site, inhibitory properties are time-dependent [50]. The hydrolyzed form of the inhibitor binds at hydrolysis due to the esterase activity of CA, and then reorient to prevent steric hindrance. Due the to this entrance of the active site, blocking substrate entry and preventing catalysis (Figure 8) [49,50]. Since required chemical transformation, coumarins are considered suicide compounds and their inhibitory coumarins bind near or within the selective pocket where the majority of unique residues are properties are time-dependent [50]. The hydrolyzed form of the inhibitor binds at the entrance of located, this class provides a strong scaffold for the design of isoform selective inhibitors. It has been the active site, blocking substrate entry and preventing catalysis (Figure 8) [49,50]. Since coumarins demonstrated that isoform specificity improves with the addition of chemical substituents to the bind near or within the selective pocket where the majority of unique residues are located, this class coumarin scaffold, with the significance of improvement dependent on the number and chemical provides scaffoldgroups for the[49]. design of isoform selective inhibitors. It has been demonstrated natureaofstrong the functional that isoform specificity improves with the addition to the coumarin Coumarin-based CAIs are commonly studied of in chemical relation tosubstituents the tumor-associated isoforms,scaffold, CA withIXthe significance of improvement dependent on the number and chemical nature of the functional and CA XII. For example, the design of 7-substituted coumarins with aryl-triazole substituents groups [49]. click chemistry led to compounds that exhibit selective inhibition in vitro in relation to utilizing Coumarin-based CAIs are commonly studied in relation to tumor-associated isoforms,that CA IX cytosolic CA Ι and CA ΙΙ [51]. Furthermore, the development ofthe 7-glycosyl coumarin inhibitors notFor only selective in vitro, but also with exhibit therapeuticsubstituents effects in mouse andshow CA XII. example, theinhibition design ofprofiles 7-substituted coumarins aryl-triazole utilizing of metastatic breast cancer the strong potentialin forvitro the in userelation of coumarin CAIs in clickmodels chemistry led to compounds thathighlight exhibit selective inhibition to cytosolic CA I with standard chemotherapies as of a novel cancer coumarin treatment [52]. andcombination CA II [51]. Furthermore, the development 7-glycosyl inhibitors that show not only

selective inhibition profiles in vitro, but also exhibit therapeutic effects in mouse models of metastatic breast cancer highlight the strong potential for the use of coumarin CAIs in combination with standard chemotherapies as a novel cancer treatment [52].

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Figure 8. (A) Trans-2-hydroxycinnamic acid (green) in complex with CA ΙΙ (gray) (PDB:5BNL) [50];

Figure 8. (A) Trans-2-hydroxycinnamic acid (green) in complex with CA II (gray) (PDB:5BNL) [50]; (B) (B) (2Z)-3-{2-hydroxy-5-[(1S)-1-hydroxy-3-methylbutyl]-4-methoxyphenyl}prop-2-enoic acid (yellow) (2Z)-3-{2-hydroxy-5-[(1S)-1-hydroxy-3-methylbutyl]-4-methoxyphenyl}prop-2-enoic acidproducts (yellow) in in complex with CA ΙΙ (gray) (PDB:3F8E) [49]; (C) Overlay of coumarin inhibitor hydrolysis Figure 8. (A) Trans-2-hydroxycinnamic acid (green) in complex with CA ΙΙ (gray) (PDB:5BNL) [50]; complex with CA II (gray) (PDB:3F8E) [49]; (C) Overlay of coumarin inhibitor hydrolysis products in in CA ΙΙ surface. (B) (2Z)-3-{2-hydroxy-5-[(1S)-1-hydroxy-3-methylbutyl]-4-methoxyphenyl}prop-2-enoic acid (yellow) CA II surface. in complex with CA ΙΙ (gray) (PDB:3F8E) [49]; (C) Overlay of coumarin inhibitor hydrolysis products 2.5. Sulfocoumarins in CA ΙΙ surface.

2.5. Sulfocoumarins While studying coumarins as lead compounds, an isostructural class, sulfocoumarins, was 2.5. Sulfocoumarins discovered to significantly inhibit CA activity; several of had a higher binding affinity to the was While studying coumarins as lead compounds, anthese isostructural class, sulfocoumarins, cancer-associated CA IX in comparison to the above coumarins [53]. As seen in previous While studying coumarins lead compounds, an of isostructural sulfocoumarins, was discovered to significantly inhibitasCA activity; several these hadclass, a higher binding affinity to coumarin-based inhibitors, the sulfocoumarin compounds are hydrolyzed by the esterase activity of discovered to significantly CA activity;toseveral of these had a higher binding the the cancer-associated CA IXinhibit in comparison the above coumarins [53]. As affinity seen intoprevious CA prior to binding. However, instead of binding outside the active site and occluding the entrance, cancer-associated CA IX insulfocoumarin comparison tocompounds the above coumarins [53]. by As the seen in previous coumarin-based inhibitors, aremolecule/hydroxide hydrolyzed esterase activity the hydrolyzed productthe anchors to the zinc-bound water ion through the of coumarin-based inhibitors, the sulfocoumarin compounds are hydrolyzed by the esterase activity of CA prior to binding. However, instead of binding outside the active site and occluding the entrance, sulfonic acid moiety (Figure 9) [53,54]. CA prior to binding. However, instead of binding outside the active site and occluding the entrance, the hydrolyzed product anchors to the zinc-bound water molecule/hydroxide ion through the sulfonic the hydrolyzed product anchors to the zinc-bound water molecule/hydroxide ion through the acid moiety (Figure 9) [53,54]. sulfonic acid moiety (Figure 9) [53,54].

Figure 9. (A) CA ΙΙ (gray) in complex with sulfocoumarin inhibitor (E)-2-(5-bromo-2-hydroxyphenyl) ethenesulfonic acid (cyan) (PDB:4BCW); (B) Surface representation of CA II in complex with sulfocoumarin inhibitor [53]. Figure 9. (A) CA ΙΙ (gray) in complex with sulfocoumarin inhibitor (E)-2-(5-bromo-2-hydroxyphenyl) Figure 9. (A) CA II (gray) in complex with sulfocoumarin inhibitor (E)-2-(5-bromo-2-hydroxyphenyl) ethenesulfonic acid (cyan) (PDB:4BCW); (B) Surface representation of CA II in complex with ethenesulfonic acid (cyan)[53]. (PDB:4BCW); (B) Surface representation of CA II in complex with sulfocoumarin inhibitor

sulfocoumarin inhibitor [53].

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The addition of chemical substituents on the heterocyclic ring, and more importantly the The of addition of chemicalgroups, substituents on the heterocyclic ring, andthe more importantly theto position position these functional has been shown to greatly affect binding affinities target of these functional groups, has been shown to greatly affect the binding affinities to target CA isoforms. CA isoforms. For example, the derivatization of the sulfocoumarin scaffold with a hydroxyl, For example, the or derivatization of thein sulfocoumarin scaffold with a hydroxyl, methane-sulfonyl methane-sulfonyl benzyloxy moiety position 6 of the 1,2-benzoxathiin 2,2-dioxide ring resulted or benzyloxy moiety in position 6 of the 1,2-benzoxathiin 2,2-dioxide ring resulted in inhibitors in inhibitors significantly more specific to CA IX, exhibiting sub-micromolar binding affinities, than significantly more specific to CA IX, exhibiting sub-micromolar binding affinities, than off-target off-target CA I or CA ΙΙ [53]. However, sulfocoumarins substituted in position 7 of this CA ringI or CA II [53]. However,for sulfocoumarins in position 7 of affinities, this ring displayed for displayed specificity CA ΙΙ with substituted low nanomolar binding which isspecificity better than CA II with low nanomolar binding affinities, which is better than acetazolamide. These 7-substituted acetazolamide. These 7-substituted sulfocoumarins therefore exhibit strong potential as a novel sulfocoumarins therefore glaucoma therapy [54]. exhibit strong potential as a novel glaucoma therapy [54]. 2.6. 2-Thioxocoumarins 2-Thioxocoumarins 2.6. Though structurally structurally related related to to the the coumarin class, aa 2-thioxocoumarin derivative was was recently recently Though coumarin class, 2-thioxocoumarin derivative observed to to bind bind in in aa region observed region of of the the CA CA II ΙΙ active active site site distinct distinct from from the the aforementioned aforementioned coumarin coumarin compounds. In complex with CA II, the inhibitor was observed to anchor to the compounds. In complex with CA ΙΙ, the inhibitor was observed to anchor to the zinc-coordinated zinc-coordinated water molecule/hydroxide ionthrough throughthe thesulfur sulfuratom atom of of the the C C == SS functionality. functionality. Additionally, Additionally, the the water molecule/hydroxide ion compound bound in its state,state, not the product as seen as for seen the previous compound boundwas wasobserved observed in original its original nothydrolysis the hydrolysis product for the coumarin inhibitors (Figure 10). The addition of the OH moiety on the 2-thioxocoumarin scaffold previous coumarin inhibitors (Figure 10). The addition of the OH moiety on the 2-thioxocoumarin increasesincreases the affinity the inhibitor the membrane-bound, cancer-associated isoforms CA IX and scaffold theofaffinity of the for inhibitor for the membrane-bound, cancer-associated isoforms CA XII several fold over off-target CA II [55]. CA IX and CA XII several fold over off-target CA ΙΙ [55].

Figure 10. (A) CA ΙΙ (gray) in complex with 6-hydroxy-2H-chromene-2-thione (green) (PDB:4WL4); Figure 10. (A) CA II (gray) in complex with 6-hydroxy-2H-chromene-2-thione (green) (PDB:4WL4); (B) Surface representation of CA II in complex with thioxocoumarin inhibitor [55]. (B) Surface representation of CA II in complex with thioxocoumarin inhibitor [55].

2.7. Fullerenes 2.7. Fullerenes Fullerenes are C60 nanoscale carbon materials that can be derivatized to achieve selectivity for targetFullerenes proteins, are suchC60 as nanoscale CA. The diameter of a fullerene scaffold is similar to to achieve that of the CA active carbon materials that can be derivatized selectivity for site, approximately nm, soThe these compounds are predicted to inhibit CAtoby occluding theactive entrance target proteins, such1as CA. diameter of a fullerene scaffold is similar that of the CA site, of substrate into the active site. However,are current structural studies fullerenethe inhibitors approximately 1 nm, so these compounds predicted to inhibit CA with by occluding entranceare of based oninto modeling and exact binding location is not yet addition of polar substrate the active site.the However, current structural studies withknown. fullereneThe inhibitors are based on substituents to the the exact fullerene scaffold has been to improve compound solubility. Additionally, modeling and binding location is notshown yet known. The addition of polar substituents to the the incorporation of amino acid ortoamine functional groups (typically common in activators) fullerene scaffold has been shown improve compound solubility. Additionally, theCA incorporation increases the number interactions with(typically CA active site residues, promotingincreases inhibition the of amino acid or amineoffunctional groups common in CA activators) thewith number bulky scaffold [56]. compounds are expected to bind to regions outside the active of interactions withAs CAfullerene active site residues, promoting inhibition with the bulky scaffold [56].site, As similar tocompounds that of coumarins, theseto inhibitors also show strong isoform-selective design. fullerene are expected bind to regions outside the potential active site,for similar to that of coumarins, these inhibitors also show strong potential for isoform-selective design. 3. Conclusions Over the last few years, there has been a significant increase in focus on the development of non-classical CAIs for the treatment of multiple diseases due to the prevalence of sulfonamide allergies among the general population. The classes of non-classical inhibitors that show strong

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3. Conclusions Over the last few years, there has been a significant increase in focus on the development of non-classical CAIs for the treatment of multiple diseases due to the prevalence of sulfonamide allergies among the general population. The classes of non-classical inhibitors that show strong potential as lead compounds for isoform-specific drug design include phenols, polyamines, carboxylic acids, and coumarins and their derivatives. As these compounds can anchor to the zinc-bound water/hydroxide ion or bind outside the active site to block substrate entry, these classes exhibit binding mechanisms atypical from the classical sulfonamide CAIs. Acknowledgments: Carrie L. Lomelino is supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under University of Florida Clinical and Translational Science Awards TL1TR001428 and UL1TR001427. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Author Contributions: Carrie L. Lomelino performed a literature search and compiled information from research articles contributed by Robert McKenna and Claudiu T. Supuran. The draft of the review was written by Carrie L. Lomelino in addition to the production of the figures. Robert McKenna and Claudiu T. Supuran reviewed the draft, made edits, and added to the text. Conflicts of Interest: The authors declare no conflict of interest.

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