**3. MAO from different species: a biochemical evaluation and a theoretical analysis using molecular simulation and a biostatistical algorithm**

As mentioned, even though amino acids lining the zMAO binding site exhibit a high level of identity with those of rat and human MAOs, a few studies have shown that the fish's en‐ zyme shows unexpected sensitivities for known specific substrates and inhibitors. Since ze‐ brafish has been proposed as a model that could be useful for the identification of novel MAO inhibitors (Kokel et al., 2010), we further characterized zMAO using three different approaches. First, we determined the inhibitory potency of a small series of compounds which have been previously evaluated against rat and human MAOs. Then, we built homol‐ ogy models of zMAO based on the crystal structures of human MAO-A or MAO-B and per‐ formed docking experiments with a drug selected from the biochemical evaluations. Finally, we used the recently described algorithm PocketMatch (Yeturu & Chandra, 2008) to explore similarities and differences between MAO isoforms from human, rat and zebrafish.

**Figure 3.** Chemical structures of the compounds used in the biochemical evaluation

Vilches-Herrera et al., 2009).

and rat MAO inhibition are from: a

**Compound**

effect

Table 1 summarizes the effects of these compounds upon zMAO and also includes, for com‐ parative purposes, the reported values of their inhibitory activities against MAO-A and -B from human and rat (Fierro et al., 2007; Hurtado-Guzmán et al., 2003; Lühr et al., 2010;

Similarities Between the Binding Sites of Monoamine Oxidase (MAO) from Different Species — Is Zebrafish a Useful

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415

MTA a NE 0.13 ± 0.02 0.25 ± 0.02 NE NE NIPAb 17.7 ± 2.6 0.48 ± 0.31 0.42 ± 0.04 >100 >100 MeONIPAb 4.8 ± 0.4 0.24 ± 0.02 0.18 ± 0.05 5.1 ± 0.4 16.3 ± 7.8 BTOc NE 10.0 ± 0.3 50.9 ± 6.1 0.46 ± 0.18 0.16 ± 0.01 ZTOc NE >100 27.5 ± 4.6 0.048 ± 0.03 0.074 ± 0.003 BTIc 30.4 ± 3.8 2.5 ± 0.2 14.1 ± 1.2 0.068 ± 0.05 0.27 ± 0.02 ZTIc NE >100 19.0 ± 0.4 0.038 ± 0.003 0.13 ±0.01

**Table 1.** zMAO inhibitory properties of known selective mammalian MAO inhibitors. Comparative data for human

The amphetamine derivative MTA, which is a potent and selective inhibitor of rat and hu‐ man MAO-A (Fierro et al., 2007; Hurtado-Guzmán et al., 2003), showed no significant effect upon zMAO activity. Similarly, the 2-arylthiomorpholine analogue ZTI, and the 2-arylthio‐ morpholin-5-one derivatives BTO and ZTO, which are highly selective MAO-B inhibitors

Hurtado-Guzmán et al., 2003; bVilches-Herrera et al 2009; c

Lühr et al, 2010. NE: No

*K***i (µM) zMAO hMAO-A rMAO-A hMAO-B rMAO-B**

### **3.1. Biochemical evaluation**

#### *3.1.1. Methods*

4-Methylthioamphetamine (MTA), 2-naphthylisopropylamine (NIPA), (6-methoxy-2-naph‐ thy)lisopropylamine (MeONIPA), all as hydrochloride salts, 2-(4'-butoxyphenyl)thiomor‐ pholine (BTI), 2-(4'-benzyloxyphenyl)thiomorpholine (ZTI), both as oxalate salts, as well as 2-(4'-butoxyphenyl)thiomorpholin-5-one (BTO) and 2-(4'-benzyloxyphenyl)thiomorpho‐ lin-5-one (ZTO) were synthesised following published methods (Hurtado-Guzmán et al., 2003; Lühr et al., 2010; Vilches-Herrera et al., 2009). The expression and purification of zMAO in *Pichia pastoris* was performed as previously described (Arslan & Edmondson, 2010). Enzyme kinetic studies were done spectrophotometrically in 50 mM potassium phos‐ phate buffer (pH = 7.4), 0.5% (w/v) reduced Triton X-100 with kynuramine as substrate. The spectrophotometer used was a Perkin-Elmer Lambda-2 UV–Vis at 25 °C.

#### *3.1.2. Results and discussion*

Figure 3 shows the chemical structures of the inhibitors evaluated.

Similarities Between the Binding Sites of Monoamine Oxidase (MAO) from Different Species — Is Zebrafish a Useful Model for the Discovery of Novel MAO Inhibitors? http://dx.doi.org/10.5772/35874 415

**Figure 3.** Chemical structures of the compounds used in the biochemical evaluation

binding energy, but natural allosteric regulations (not always considered) might not favor such conformations; protein structures from databases could have been determined in dif‐ ferent conformational states (active, inactive, closed, open, etc.); finally, it is also very impor‐

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Beyond these considerations, the continuous increase in both the number of protein struc‐ tures and computational power, augurs the development of ever more accurate similarity searching tools, which likely will allow not only better results in virtual screening programs

**3. MAO from different species: a biochemical evaluation and a theoretical**

As mentioned, even though amino acids lining the zMAO binding site exhibit a high level of identity with those of rat and human MAOs, a few studies have shown that the fish's en‐ zyme shows unexpected sensitivities for known specific substrates and inhibitors. Since ze‐ brafish has been proposed as a model that could be useful for the identification of novel MAO inhibitors (Kokel et al., 2010), we further characterized zMAO using three different approaches. First, we determined the inhibitory potency of a small series of compounds which have been previously evaluated against rat and human MAOs. Then, we built homol‐ ogy models of zMAO based on the crystal structures of human MAO-A or MAO-B and per‐ formed docking experiments with a drug selected from the biochemical evaluations. Finally, we used the recently described algorithm PocketMatch (Yeturu & Chandra, 2008) to explore

**analysis using molecular simulation and a biostatistical algorithm**

similarities and differences between MAO isoforms from human, rat and zebrafish.

spectrophotometer used was a Perkin-Elmer Lambda-2 UV–Vis at 25 °C.

Figure 3 shows the chemical structures of the inhibitors evaluated.

4-Methylthioamphetamine (MTA), 2-naphthylisopropylamine (NIPA), (6-methoxy-2-naph‐ thy)lisopropylamine (MeONIPA), all as hydrochloride salts, 2-(4'-butoxyphenyl)thiomor‐ pholine (BTI), 2-(4'-benzyloxyphenyl)thiomorpholine (ZTI), both as oxalate salts, as well as 2-(4'-butoxyphenyl)thiomorpholin-5-one (BTO) and 2-(4'-benzyloxyphenyl)thiomorpho‐ lin-5-one (ZTO) were synthesised following published methods (Hurtado-Guzmán et al., 2003; Lühr et al., 2010; Vilches-Herrera et al., 2009). The expression and purification of zMAO in *Pichia pastoris* was performed as previously described (Arslan & Edmondson, 2010). Enzyme kinetic studies were done spectrophotometrically in 50 mM potassium phos‐ phate buffer (pH = 7.4), 0.5% (w/v) reduced Triton X-100 with kynuramine as substrate. The

**3.1. Biochemical evaluation**

*3.1.2. Results and discussion*

*3.1.1. Methods*

Applications

414

tant to consider the solvent and ion concentrations in every system.

but also a novel view on the evolution of structure and function of proteins.

Table 1 summarizes the effects of these compounds upon zMAO and also includes, for com‐ parative purposes, the reported values of their inhibitory activities against MAO-A and -B from human and rat (Fierro et al., 2007; Hurtado-Guzmán et al., 2003; Lühr et al., 2010; Vilches-Herrera et al., 2009).


**Table 1.** zMAO inhibitory properties of known selective mammalian MAO inhibitors. Comparative data for human and rat MAO inhibition are from: a Hurtado-Guzmán et al., 2003; bVilches-Herrera et al 2009; c Lühr et al, 2010. NE: No effect

The amphetamine derivative MTA, which is a potent and selective inhibitor of rat and hu‐ man MAO-A (Fierro et al., 2007; Hurtado-Guzmán et al., 2003), showed no significant effect upon zMAO activity. Similarly, the 2-arylthiomorpholine analogue ZTI, and the 2-arylthio‐ morpholin-5-one derivatives BTO and ZTO, which are highly selective MAO-B inhibitors

(Lühr et al., 2010), did not inhibit the fish's enzyme. In contrast, naphthylisopropylamine de‐ rivatives NIPA and MeONIPA, which are selective inhibitors of MAO-A (Vilches-Herrera et al., 2009), as well as the 2-arylthiomorpholine derivative BTI which selectively inhibits MAO-B (Lühr et al., 2010), exhibited zMAO inhibitory properties with *K*<sup>i</sup> values in the mi‐ cromolar range. MeONIPA was the most potent compound of the series evaluated, showing a *K*<sup>i</sup> value (4.8 μM) very similar to that found against human MAO-B (5.1 μM). These results agree with a notion that can be inferred from previous data (Aldeco et al., 2011; Anichtchik et al., 2006), indicating that effects on zMAO cannot be straightforwardly used to predict an effect upon either MAO-A or MAO-B. In addition, these data suggest that the zMAO bind‐ ing site is significantly different from those of both MAO-A and MAO-B from mammals.

(*S*)-amphetamine derivatives (which are always dextrorotatory) are usually the eutomers at MAO (Hurtado-Guzmán et al., 2003). All other docking conditions were as previously re‐ ported (Fierro et al., 2007; Vilches-Herrera et al., 2009). Briefly, the grid maps were calculat‐ ed using the autogrid4 option and were centered on the putative ligand-binding site. The volumes chosen for the grid maps were made up of 40 × 40 × 40 points, with a grid-point spacing of 0.375 Å. The autotors option was used to define the rotating bond in the ligand. The docked compound complexes were built using the lowest docked-energy binding posi‐ tions. MeONIPA was built using Gaussian03 (Frisch et al., 2004) and the partial charges

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417

Figure 4 depicts the global zMAO models obtained using human MAO-A (left) and human MAO-B (right) as templates. As expected, the overall structure of zMAO was similar to those of the human enzymes. The presumed ligand binding site appears lined by a series of hydrophobic residues and the isoalloxazine ring of the flavin cofactor (top inset Fig. 4). Ami‐ no acids forming the binding site of zMAO and human MAO-A and -B are shown in insets

**Figure 4.** Cartoons of zMAO models obtained using human MAO-A (left) or human MAO-B (right). Insets show the main amino acids of the active sites of zMAO (top), human MAO-A (left) and human MAO-B (right). Amino acids in

white, green or blue indicate apolar, polar or positively charged residues respectively.

were corrected using ESP methodology.

*3.2.3. Results and discussion*

of Figure 4.

#### **3.2. Homology models of zMAO and molecular docking**

### *3.2.1. Modeling methods*

Since neither the MAO-A nor MAO-B structure can be chosen *a priori* as a better template for modeling zMAO, we decided to build two different models using each isoform of human MAO as templates. The MAO-A (Protein Data Bank, PDB code: 2BXS) and MAO-B (PDB code: 2BYB) crystal structures at 3.15 Å and 2.2 Å resolution respectively (De Colibus et al., 2005) were employed. The amino acid sequence and crystal structure of each protein were extracted from the National Center for Biotechnology Information (NCBI) and PDB databas‐ es. Sequence alignments were prepared separately. Models were built using standard pa‐ rameters and the outcomes were ranked on the basis of the internal scoring function of the program MODELLER9v6 (Sali & Blundell, 1993). The best model obtained in each case (us‐ ing MAO-A or MAO-B as template) was submitted to the H++ server (Gordon et al., 2005; http://biophysics.cs.vt.edu/H++) to compute p*K*<sup>a</sup> values of ionizable groups and to add miss‐ ing hydrogen atoms according to the specified pH of the environment. Each structure select‐ ed was inserted into a POPC membrane, TIP3 solvated and ions were added creating an overall neutral system simulating approximately 0.2 M NaCl. The ions were equally distrib‐ uted in a water box. The final system was subjected to a molecular dynamics (MD) simula‐ tion for 5 ns using NAMD 2.6 (Phillips et al., 2005). The NPT ensemble was used to perform MD calculations. Periodic boundary conditions were applied to the system in the three coor‐ dinate directions. A pressure of 1 atm was used and temperature was kept at 310 K. The simulation time was sufficient to obtain an equilibrated system (RMSD < 2 Å). Stereochemi‐ cal and energy quality of the homology models were evaluated using the PROSAII server (Wiederstain & Sippl 2007) and Procheck (Laskowski et al., 1993)

#### *3.2.2. Docking methods*

Dockings of (*S*)-MeONIPA in the zMAO models, as well as in the human MAO-A and MAO-B structures were done using the AutoDock 4.0 suite (Morris et al., 1998). MeONIPA was selected for this study since it was the most potent zMAO inhibitor of the series evaluat‐ ed and because it also inhibited both human MAO-A and MAO-B at low concentrations. The choice of the (*S*)-isomer for MeONIPA docking experiments was done on the basis that (*S*)-amphetamine derivatives (which are always dextrorotatory) are usually the eutomers at MAO (Hurtado-Guzmán et al., 2003). All other docking conditions were as previously re‐ ported (Fierro et al., 2007; Vilches-Herrera et al., 2009). Briefly, the grid maps were calculat‐ ed using the autogrid4 option and were centered on the putative ligand-binding site. The volumes chosen for the grid maps were made up of 40 × 40 × 40 points, with a grid-point spacing of 0.375 Å. The autotors option was used to define the rotating bond in the ligand. The docked compound complexes were built using the lowest docked-energy binding posi‐ tions. MeONIPA was built using Gaussian03 (Frisch et al., 2004) and the partial charges were corrected using ESP methodology.

### *3.2.3. Results and discussion*

(Lühr et al., 2010), did not inhibit the fish's enzyme. In contrast, naphthylisopropylamine de‐ rivatives NIPA and MeONIPA, which are selective inhibitors of MAO-A (Vilches-Herrera et al., 2009), as well as the 2-arylthiomorpholine derivative BTI which selectively inhibits MAO-B (Lühr et al., 2010), exhibited zMAO inhibitory properties with *K*<sup>i</sup> values in the mi‐ cromolar range. MeONIPA was the most potent compound of the series evaluated, showing a *K*<sup>i</sup> value (4.8 μM) very similar to that found against human MAO-B (5.1 μM). These results agree with a notion that can be inferred from previous data (Aldeco et al., 2011; Anichtchik et al., 2006), indicating that effects on zMAO cannot be straightforwardly used to predict an effect upon either MAO-A or MAO-B. In addition, these data suggest that the zMAO bind‐ ing site is significantly different from those of both MAO-A and MAO-B from mammals.

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Since neither the MAO-A nor MAO-B structure can be chosen *a priori* as a better template for modeling zMAO, we decided to build two different models using each isoform of human MAO as templates. The MAO-A (Protein Data Bank, PDB code: 2BXS) and MAO-B (PDB code: 2BYB) crystal structures at 3.15 Å and 2.2 Å resolution respectively (De Colibus et al., 2005) were employed. The amino acid sequence and crystal structure of each protein were extracted from the National Center for Biotechnology Information (NCBI) and PDB databas‐ es. Sequence alignments were prepared separately. Models were built using standard pa‐ rameters and the outcomes were ranked on the basis of the internal scoring function of the program MODELLER9v6 (Sali & Blundell, 1993). The best model obtained in each case (us‐ ing MAO-A or MAO-B as template) was submitted to the H++ server (Gordon et al., 2005; http://biophysics.cs.vt.edu/H++) to compute p*K*<sup>a</sup> values of ionizable groups and to add miss‐ ing hydrogen atoms according to the specified pH of the environment. Each structure select‐ ed was inserted into a POPC membrane, TIP3 solvated and ions were added creating an overall neutral system simulating approximately 0.2 M NaCl. The ions were equally distrib‐ uted in a water box. The final system was subjected to a molecular dynamics (MD) simula‐ tion for 5 ns using NAMD 2.6 (Phillips et al., 2005). The NPT ensemble was used to perform MD calculations. Periodic boundary conditions were applied to the system in the three coor‐ dinate directions. A pressure of 1 atm was used and temperature was kept at 310 K. The simulation time was sufficient to obtain an equilibrated system (RMSD < 2 Å). Stereochemi‐ cal and energy quality of the homology models were evaluated using the PROSAII server

Dockings of (*S*)-MeONIPA in the zMAO models, as well as in the human MAO-A and MAO-B structures were done using the AutoDock 4.0 suite (Morris et al., 1998). MeONIPA was selected for this study since it was the most potent zMAO inhibitor of the series evaluat‐ ed and because it also inhibited both human MAO-A and MAO-B at low concentrations. The choice of the (*S*)-isomer for MeONIPA docking experiments was done on the basis that

**3.2. Homology models of zMAO and molecular docking**

(Wiederstain & Sippl 2007) and Procheck (Laskowski et al., 1993)

*3.2.1. Modeling methods*

Applications

416

*3.2.2. Docking methods*

Figure 4 depicts the global zMAO models obtained using human MAO-A (left) and human MAO-B (right) as templates. As expected, the overall structure of zMAO was similar to those of the human enzymes. The presumed ligand binding site appears lined by a series of hydrophobic residues and the isoalloxazine ring of the flavin cofactor (top inset Fig. 4). Ami‐ no acids forming the binding site of zMAO and human MAO-A and -B are shown in insets of Figure 4.

**Figure 4.** Cartoons of zMAO models obtained using human MAO-A (left) or human MAO-B (right). Insets show the main amino acids of the active sites of zMAO (top), human MAO-A (left) and human MAO-B (right). Amino acids in white, green or blue indicate apolar, polar or positively charged residues respectively.

As shown in Figure 5, docking experiments revealed that in both zMAO models, MeONIPA exhibits a binding mode where the aromatic ring is oriented almost perpendicularly to the isoalloxazine ring of FAD, with the methoxyl group pointing to the binding site entrance, whereas the aminopropyl chain points toward the isoalloxazine ring and appears positioned close to two tyrosine residues which, together with the isoalloxazine ring, form the so-called aromatic cage (Figs. 5 A and 5B). Interestingly, docking of MeONIPA in both human MAO-A and MAO-B, yielded binding modes where the inhibitor molecule adopted an almost op‐ posite orientation to those observed in zMAO models. Thus, the most energetically favorable conformations of MeONIPA were those in which the amino group points away from the flavin ring, whereas the methoxyl group is located between the corresponding ty‐ rosine residues (Figs. 5 C and 5D). These results suggest that the different inhibitory poten‐ cies of MeONIPA (and likely other inhibitors) toward zebrafish and human MAOs, might be attributed to the differential binding modes exhibited by the drug. Similar conclusions at‐ tempting to explain why MAO inhibitors show differential inhibition properties upon MAO from different species have been reached in previous studies (Fierro et al., 2007; Nandinga‐ ma et al., 2002). Moreover, our findings suggest that, even in the cases where similar poten‐ cies are detected, the mechanism of enzyme inhibition for a given drug might be different in zebrafish and human MAOs.

**3.3. Similarities between the binding sites of MAO from different species.**

The structures of human and rat MAO-A co-crystallized with clorgyline (PDB codes: 2BXS and 1O5W respectively) and human MAO-B co-crystallized with *l*-deprenyl (PDB code: 2BYB) were employed. Furthermore, structures of zMAO models and human MAO-A and MAO-B obtained after docking of MeONIPA (see previous section), were used in additional

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The PocketMatch algorithm was selected for this study due to its relatively low computa‐ tional complexity and high performance. All aspects involved in binding site comparisons followed the procedure published in the original article describing the algorithm (Yeturu & Chandra, 2008). Briefly, each binding site was considered as that determined by the residues for which one or more atoms surround either a crystallographic or a docked ligand at a giv‐ en distance (4 Å by default; in some cases distances from 3 Å to 10 Å from the ligand were considered; see following section). Each residue was classified into one of 5 groups, taken into account its chemical properties. Then, each residue was represented as a set of three points corresponding to the coordinates of the C-Alpha, the C-Beta and the Centroid Atom of the side chain. Distances between every three points of each residue in the binding sites were measured. All distances computed were sorted in ascending order and stored in sets of distances organized by type of pairs of points and type of pairs of tags. The sorted and or‐ ganized distances were aligned and compared using a threshold of 0.5 Å, which was estab‐ lished considering the natural dynamics of biological systems. The similarity between sites, referred to as the PMScore, was measured by scoring the alignment of the pair of sites under comparison. Thus, the PMScore represents the percentage of the number of "matches" cal‐ culated over the maximal number of distances computed for each binding site. A PMScore of 0.5 (50 %) or higher was considered as indicative of similarity between binding sites.

Initially, we compared human and rat MAO-A. The amino acid sequence in the active sites of both proteins is identical, and therefore we expected to find a high degree of similarity. Surprisingly, a PMScore value of 0.27 was obtained after comparing the residues located at 4 Å from the ligand (clorgyline in both proteins), which is the PocketMatch default condition. It should be considered that PMScores > 0.5 are indicative of binding site similarity, whereas values below 0.5 indicate lack of similarity. It should also be noted that, as shown in the original report by Yeturu & Chandra (2008), a distance of 4 Å from the ligand was clearly suitable to find similarities between a series of structurally related and unrelated proteins. Therefore, it was rather intriguing that such a low PMScore should be obtained, suggesting the existence of relevant differences between rat and human MAO-A binding sites, most likely in the form in which residues in close proximity to the ligand are arranged. Such a conformational difference has been revealed by the crystal structures of both proteins,

*3.3.1. Protein structures employed*

*3.3.2. Binding site comparison methods*

*3.3.3. Results and discussion*

comparisons.

**Figure 5.** Comparison of the binding modes of MeONIPA into zMAO (A and B), human MAO-A (C) and human MAO-B (D) active sites. Figures 5 A and 5Bshow the docking poses of MeONIPA into zMAO models obtained using human MAO-A and human MAO-B respectively. Main active site amino acid residues and FAD are rendered as stick models.

### **3.3. Similarities between the binding sites of MAO from different species.**

#### *3.3.1. Protein structures employed*

As shown in Figure 5, docking experiments revealed that in both zMAO models, MeONIPA exhibits a binding mode where the aromatic ring is oriented almost perpendicularly to the isoalloxazine ring of FAD, with the methoxyl group pointing to the binding site entrance, whereas the aminopropyl chain points toward the isoalloxazine ring and appears positioned close to two tyrosine residues which, together with the isoalloxazine ring, form the so-called aromatic cage (Figs. 5 A and 5B). Interestingly, docking of MeONIPA in both human MAO-A and MAO-B, yielded binding modes where the inhibitor molecule adopted an almost op‐ posite orientation to those observed in zMAO models. Thus, the most energetically favorable conformations of MeONIPA were those in which the amino group points away from the flavin ring, whereas the methoxyl group is located between the corresponding ty‐ rosine residues (Figs. 5 C and 5D). These results suggest that the different inhibitory poten‐ cies of MeONIPA (and likely other inhibitors) toward zebrafish and human MAOs, might be attributed to the differential binding modes exhibited by the drug. Similar conclusions at‐ tempting to explain why MAO inhibitors show differential inhibition properties upon MAO from different species have been reached in previous studies (Fierro et al., 2007; Nandinga‐ ma et al., 2002). Moreover, our findings suggest that, even in the cases where similar poten‐ cies are detected, the mechanism of enzyme inhibition for a given drug might be different in

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

**Figure 5.** Comparison of the binding modes of MeONIPA into zMAO (A and B), human MAO-A (C) and human MAO-B (D) active sites. Figures 5 A and 5Bshow the docking poses of MeONIPA into zMAO models obtained using human MAO-A and human MAO-B respectively. Main active site amino acid residues and FAD are rendered as stick models.

zebrafish and human MAOs.

Applications

418

The structures of human and rat MAO-A co-crystallized with clorgyline (PDB codes: 2BXS and 1O5W respectively) and human MAO-B co-crystallized with *l*-deprenyl (PDB code: 2BYB) were employed. Furthermore, structures of zMAO models and human MAO-A and MAO-B obtained after docking of MeONIPA (see previous section), were used in additional comparisons.

### *3.3.2. Binding site comparison methods*

The PocketMatch algorithm was selected for this study due to its relatively low computa‐ tional complexity and high performance. All aspects involved in binding site comparisons followed the procedure published in the original article describing the algorithm (Yeturu & Chandra, 2008). Briefly, each binding site was considered as that determined by the residues for which one or more atoms surround either a crystallographic or a docked ligand at a giv‐ en distance (4 Å by default; in some cases distances from 3 Å to 10 Å from the ligand were considered; see following section). Each residue was classified into one of 5 groups, taken into account its chemical properties. Then, each residue was represented as a set of three points corresponding to the coordinates of the C-Alpha, the C-Beta and the Centroid Atom of the side chain. Distances between every three points of each residue in the binding sites were measured. All distances computed were sorted in ascending order and stored in sets of distances organized by type of pairs of points and type of pairs of tags. The sorted and or‐ ganized distances were aligned and compared using a threshold of 0.5 Å, which was estab‐ lished considering the natural dynamics of biological systems. The similarity between sites, referred to as the PMScore, was measured by scoring the alignment of the pair of sites under comparison. Thus, the PMScore represents the percentage of the number of "matches" cal‐ culated over the maximal number of distances computed for each binding site. A PMScore of 0.5 (50 %) or higher was considered as indicative of similarity between binding sites.

#### *3.3.3. Results and discussion*

Initially, we compared human and rat MAO-A. The amino acid sequence in the active sites of both proteins is identical, and therefore we expected to find a high degree of similarity. Surprisingly, a PMScore value of 0.27 was obtained after comparing the residues located at 4 Å from the ligand (clorgyline in both proteins), which is the PocketMatch default condition. It should be considered that PMScores > 0.5 are indicative of binding site similarity, whereas values below 0.5 indicate lack of similarity. It should also be noted that, as shown in the original report by Yeturu & Chandra (2008), a distance of 4 Å from the ligand was clearly suitable to find similarities between a series of structurally related and unrelated proteins. Therefore, it was rather intriguing that such a low PMScore should be obtained, suggesting the existence of relevant differences between rat and human MAO-A binding sites, most likely in the form in which residues in close proximity to the ligand are arranged. Such a conformational difference has been revealed by the crystal structures of both proteins,

which show that the cavity-shaping loop 210–216 and specifically residues Gln215 and Glu216 are differentially oriented in human and rat MAO-A (De Colibus et al., 2005). This differential arrangement determines a larger volume of the active site of human MAO-A (550 Å3 ) as compared to that of rat MAO-A (450 Å3 ). Thus, our results confirm that rat and human MAOs are not as similar as could be inferred from the analysis of their amino acid sequences, and highlight the sensitivity of PocketMatch to determine subtle differences be‐ tween highly related proteins.

Despite these considerations, we developed a script that allows the automatic evaluation of PMScores considering distances from 3 Å to 10 Å from the ligand, with the hope that such an analysis could yield further information regarding the similarity of the binding sites of MAOs. Thus, we were able to build "similarity profiles", which graphically show at what distance from the ligand (if any) the binding sites begin to be similar. Figure 6 shows the similarity profile after comparing rat and human MAO-A.

> **Figure 7.** Similarity profile between human MAO-A (co-crystalized with clorgyline) and human MAO-B (co-crystalized with deprenyl), as calculated using PocketMatch. The horizontal black line indicates PMScore = 0.5. The vertical black line indicates the distance from the ligand where the PMScore begins to be consistently greater than 0.5. Each point

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As shown in Figures 8C and 8D, at a distance of 6.4 Å from the ligand, several amino acids

**Figure 8.** Binding site residues surrounding the inhibitors clorgyline (blue) and deprenyl (pink) bound to human MAO-A (HMAO-A), rat MAO-A (RMAO-A) or human MAO-B (HMAO-B). Figures 8A and 8B show the residues located at 4.5

Å from the ligand, while figures 8C and 8D show the residues located at 6.5 Å from the ligand

considered in the similarity determination are located outside the binding site.

corresponds to the PMScore.

**Figure 6.** Similarity profile between rat and human MAO-A, both co-crystalized with clorgyline, as calculated using PocketMatch. The horizontal black line indicates PMScore = 0.5. The vertical black line indicates the distance from the ligand where the PMScore begins to be consistently greater than 0.5. Each point corresponds to the PMScore.

As can be seen, PMScores greater than 0.5 appeared at 4.5 Å and were consistently observed at longer distances from the ligand. Since most amino acids located at 4.5 Å from the ligand line the binding site (see Figure 8A and 8B), these results indicate that, beyond the shape dif‐ ferences revealed by crystal structures and detected by PocketMatch, the binding sites of MAO-A from rat and human are quite similar.

In contrast, when binding sites of human MAO-A and MAO-B were compared, PMScores indicating similarity (> 0.5) were only found at distances higher than 6.4 Å from the ligand (Fig. 7).

Similarities Between the Binding Sites of Monoamine Oxidase (MAO) from Different Species — Is Zebrafish a Useful Model for the Discovery of Novel MAO Inhibitors? http://dx.doi.org/10.5772/35874 421

which show that the cavity-shaping loop 210–216 and specifically residues Gln215 and Glu216 are differentially oriented in human and rat MAO-A (De Colibus et al., 2005). This differential arrangement determines a larger volume of the active site of human MAO-A

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

human MAOs are not as similar as could be inferred from the analysis of their amino acid sequences, and highlight the sensitivity of PocketMatch to determine subtle differences be‐

Despite these considerations, we developed a script that allows the automatic evaluation of PMScores considering distances from 3 Å to 10 Å from the ligand, with the hope that such an analysis could yield further information regarding the similarity of the binding sites of MAOs. Thus, we were able to build "similarity profiles", which graphically show at what distance from the ligand (if any) the binding sites begin to be similar. Figure 6 shows the

**Figure 6.** Similarity profile between rat and human MAO-A, both co-crystalized with clorgyline, as calculated using PocketMatch. The horizontal black line indicates PMScore = 0.5. The vertical black line indicates the distance from the ligand where the PMScore begins to be consistently greater than 0.5. Each point corresponds to the PMScore.

As can be seen, PMScores greater than 0.5 appeared at 4.5 Å and were consistently observed at longer distances from the ligand. Since most amino acids located at 4.5 Å from the ligand line the binding site (see Figure 8A and 8B), these results indicate that, beyond the shape dif‐ ferences revealed by crystal structures and detected by PocketMatch, the binding sites of

In contrast, when binding sites of human MAO-A and MAO-B were compared, PMScores indicating similarity (> 0.5) were only found at distances higher than 6.4 Å from the ligand

). Thus, our results confirm that rat and

) as compared to that of rat MAO-A (450 Å3

similarity profile after comparing rat and human MAO-A.

MAO-A from rat and human are quite similar.

(Fig. 7).

(550 Å3

Applications

420

tween highly related proteins.

**Figure 7.** Similarity profile between human MAO-A (co-crystalized with clorgyline) and human MAO-B (co-crystalized with deprenyl), as calculated using PocketMatch. The horizontal black line indicates PMScore = 0.5. The vertical black line indicates the distance from the ligand where the PMScore begins to be consistently greater than 0.5. Each point corresponds to the PMScore.

As shown in Figures 8C and 8D, at a distance of 6.4 Å from the ligand, several amino acids considered in the similarity determination are located outside the binding site.

**Figure 8.** Binding site residues surrounding the inhibitors clorgyline (blue) and deprenyl (pink) bound to human MAO-A (HMAO-A), rat MAO-A (RMAO-A) or human MAO-B (HMAO-B). Figures 8A and 8B show the residues located at 4.5 Å from the ligand, while figures 8C and 8D show the residues located at 6.5 Å from the ligand

Therefore, the similarity profile shown in Figure 7 indicates that human MAO-A and MAO-B binding sites are less similar than those of rat and human MAO-A. It also shows that, al‐ though showing differences at their binding sites, human MAO-A and MAO-B exhibit a high degree of global structural similarity (all PMScores obtained at distances longer than 6.5 Å were well over 0.5). Though both findings might be considered obvious from the anal‐ ysis of each protein sequence and function, they confirm the suitability of PocketMatch to find and predict such characteristics, an aspect that could be particularly useful when com‐ paring proteins from which less functional information is available. In addition, our results suggest that in some cases the determination of similarity profiles can be more informative than point comparisons.

Figures 9 and 10 show the similarity profiles after comparing the homology models of zMAO with those of human MAO-A and MAO-B, respectively. As mentioned, in all cases, MeONIPA docked in each MAO structure was used as ligand.

cal nature of the residues forming the site (Yeturu & Chandra, 2008), these two factors are likely involved in the differences detected between the MAO isoforms. Considering the se‐ quence identity between zebrafish and human enzymes, one may predict that conformation‐ al differences are more important when comparing zMAO and human MAO-A, while the chemical features of the residues are more relevant to the differences between zMAO and human MAO-B. Nevertheless, further analyses are necessary to determine the relative con‐

**Figure 10.** Similarity profile between zMAO (in this case the model corresponds to that based on human MAO-B) and human MAO-B, as calculated using PocketMatch. In both proteins, docked MeONIPA was used as ligand. The horizon‐ tal black line indicates PMScore = 0.5. The vertical black line indicates the distance from the ligand where the PMScore

Similarities Between the Binding Sites of Monoamine Oxidase (MAO) from Different Species — Is Zebrafish a Useful

Model for the Discovery of Novel MAO Inhibitors?

http://dx.doi.org/10.5772/35874

423

In summary, results from biochemical evaluation, molecular simulation and similarity de‐ tection studies presented here add novel evidence to the notion that even though zMAO ex‐ hibits some functional and structural properties overlapping those of MAO-A and -B, the zebrafish protein behaves quite distinctively from its mammalian counterparts. Therefore, although still an attractive model for drug discovery, in our opinion zebrafish is not a useful

We thank Dr. K. Yeturu and Prof. N. Chandra for their valuable comments regarding Pocket Match results and functioning. We also thank Prof. Bruce K. Cassels for critical reading of the

model for the identification of novel MAO inhibitors aimed for use in humans.

tribution of each aspect to the differences found.

begins to be consistently greater than 0.5. Each point corresponds to the PMScore.

**4. Conclusion**

**Acknowledgements**

**Figure 9.** Similarity profile between zMAO (in this case the model corresponds to that based on human MAO-A) and human MAO-A, as calculated using PocketMatch. In both proteins, docked MeONIPA was used as ligand. The horizon‐ tal black line indicates PMScore = 0.5. The vertical black line indicates the distance from the ligand where the PMScore begins to be consistently greater than 0.5. Each point corresponds to the PMScore.

As shown in Figures 9 and 10, PMScores indicative of similarity between the binding sites of zMAO and human MAO-A or MAO-B (i.e., PMScore > 0.5) were consistently seen at distan‐ ces higher than 6 Å from the ligand. It should be noted that comparable values were ob‐ tained even though the zMAO model was built using either human MAO-A or MAO-B as templates, and regardless of which human enzyme was used for the comparison. These re‐ sults suggest that the zMAO binding site is as different from those of both human isoforms as the binding site of MAO-A differs from that of MAO-B. In addition, the similarity profiles of zMAO against both human proteins indicate that global structural similarity is found across these species, while the main differences are found at their binding sites. Since, to perform the similarity determination, PocketMatch considers both the shape and the chemi‐

Similarities Between the Binding Sites of Monoamine Oxidase (MAO) from Different Species — Is Zebrafish a Useful Model for the Discovery of Novel MAO Inhibitors? http://dx.doi.org/10.5772/35874 423

**Figure 10.** Similarity profile between zMAO (in this case the model corresponds to that based on human MAO-B) and human MAO-B, as calculated using PocketMatch. In both proteins, docked MeONIPA was used as ligand. The horizon‐ tal black line indicates PMScore = 0.5. The vertical black line indicates the distance from the ligand where the PMScore begins to be consistently greater than 0.5. Each point corresponds to the PMScore.

cal nature of the residues forming the site (Yeturu & Chandra, 2008), these two factors are likely involved in the differences detected between the MAO isoforms. Considering the se‐ quence identity between zebrafish and human enzymes, one may predict that conformation‐ al differences are more important when comparing zMAO and human MAO-A, while the chemical features of the residues are more relevant to the differences between zMAO and human MAO-B. Nevertheless, further analyses are necessary to determine the relative con‐ tribution of each aspect to the differences found.

### **4. Conclusion**

Therefore, the similarity profile shown in Figure 7 indicates that human MAO-A and MAO-B binding sites are less similar than those of rat and human MAO-A. It also shows that, al‐ though showing differences at their binding sites, human MAO-A and MAO-B exhibit a high degree of global structural similarity (all PMScores obtained at distances longer than 6.5 Å were well over 0.5). Though both findings might be considered obvious from the anal‐ ysis of each protein sequence and function, they confirm the suitability of PocketMatch to find and predict such characteristics, an aspect that could be particularly useful when com‐ paring proteins from which less functional information is available. In addition, our results suggest that in some cases the determination of similarity profiles can be more informative

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Figures 9 and 10 show the similarity profiles after comparing the homology models of zMAO with those of human MAO-A and MAO-B, respectively. As mentioned, in all cases,

**Figure 9.** Similarity profile between zMAO (in this case the model corresponds to that based on human MAO-A) and human MAO-A, as calculated using PocketMatch. In both proteins, docked MeONIPA was used as ligand. The horizon‐ tal black line indicates PMScore = 0.5. The vertical black line indicates the distance from the ligand where the PMScore

As shown in Figures 9 and 10, PMScores indicative of similarity between the binding sites of zMAO and human MAO-A or MAO-B (i.e., PMScore > 0.5) were consistently seen at distan‐ ces higher than 6 Å from the ligand. It should be noted that comparable values were ob‐ tained even though the zMAO model was built using either human MAO-A or MAO-B as templates, and regardless of which human enzyme was used for the comparison. These re‐ sults suggest that the zMAO binding site is as different from those of both human isoforms as the binding site of MAO-A differs from that of MAO-B. In addition, the similarity profiles of zMAO against both human proteins indicate that global structural similarity is found across these species, while the main differences are found at their binding sites. Since, to perform the similarity determination, PocketMatch considers both the shape and the chemi‐

MeONIPA docked in each MAO structure was used as ligand.

begins to be consistently greater than 0.5. Each point corresponds to the PMScore.

than point comparisons.

Applications

422

In summary, results from biochemical evaluation, molecular simulation and similarity de‐ tection studies presented here add novel evidence to the notion that even though zMAO ex‐ hibits some functional and structural properties overlapping those of MAO-A and -B, the zebrafish protein behaves quite distinctively from its mammalian counterparts. Therefore, although still an attractive model for drug discovery, in our opinion zebrafish is not a useful model for the identification of novel MAO inhibitors aimed for use in humans.

### **Acknowledgements**

We thank Dr. K. Yeturu and Prof. N. Chandra for their valuable comments regarding Pocket Match results and functioning. We also thank Prof. Bruce K. Cassels for critical reading of the

manuscript. This work was funded by MSI Grant P05/001-F, PBCT grant PDA-23 to AF and FONDECYT Grants 110-85002 to AF, 110-0542 to PI-V and 109-0037 to MR-P. D.E.E. acknowl‐ edges research support from the National Institutes of Health GM 29433

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Model for the Discovery of Novel MAO Inhibitors?

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