**1. Introduction**

Zebrafish (*Danio rerio*) is an animal model that is attracting increasing interest in pharmacol‐ ogy and toxicology. The relatively ease with which large numbers of individuals can be ob‐ tained and their inexpensive maintenance makes zebrafish a particularly suitable tool for drug discovery. Thus, in recent years diverse compounds have been assayed both in larval and adult specimens and changes of behavioral patterns, for instance, have been related to anxiolytic, addictive or cognitive effects. In this context, the molecular characterization of drug targets in zebrafish, comparing them to their mammalian counterparts, arises as a sub‐ ject of paramount importance.

Monoamine oxidase (MAO) is the main catabolic enzyme of monoamine neurotransmitters and the primary target of several clinically relevant antidepressant and antiparkinsonian drugs. In mammals, it exists in two isoforms termed MAO-A and MAO-B, which share a number of structural and mechanistic features, but differ in genetic origin, tissue localization and inhibitor selectivity. High-resolution structures of MAOs from rat and human have

© 2013 Fierro et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Fierro et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

been reported during the last decade, allowing detailed comparison of their overall struc‐ tures and respective active sites. On the other hand, a few studies have shown that zebrafish contains a single MAO gene and that enzyme activity is due to a single form (zMAO) which resembles, but is distinct from, both mammalian MAO-A and MAO-B. No three-dimension‐ al structural data exist thus far for zMAO. Sequence comparison of the putative substrate binding site of zMAO with those of human MAO isoforms suggests that the fish enzyme re‐ sembles mammalian MAO-A more than MAO-B. Nevertheless, biochemical studies have shown that zMAO exhibits such unique behavior toward MAO-A and -B substrates and in‐ hibitors, that the results of studies using zebrafish MAO function, either as a disease model or for drug screening, should be considered with caution.

**2. Zebrafish as a model in pharmacology, monoamine oxidase and**

**2.1. Zebrafish as an animal model in pharmacology and neurobehavioral studies**

2005).

**computational methods to evaluate binding site similarities: An overview**

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

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In order to understand complex behaviors observed in nature, scientists have always tried to develop models that could be used and tested under controlled conditions in the labora‐ tory. In the last 30 years a new animal model, zebrafish (*Danio rerio*), has emerged as a pow‐ erful tool mostly for studying developmental biology. The scientific potential of zebrafish was originally assessed by George Streisinger (Streisinger et al., 1981). This work was the starting point for rapid progress in molecular and genetic analysis of zebrafish neurodevel‐ opment, which allowed the construction of many genetic mutants and the identification of several genes that affect different brain functions such as learning and memory (Norton & Bally-Cuif, 2010). During the last decade zebrafish has also become an attractive model for behavioral and drug discovery studies, particularly those related to actions in the central nervous systems (Chakraborty & Hsu, 2009; King, 2009; Rubinstein, 2006; Zon & Peterson,

Zebrafish develop rapidly and almost all organs are developed at 7 days post-fertilization. Their fecundity makes it easy to obtain large numbers of individuals for experimentation, which are relatively inexpensive to maintain. In addition, they can absorb chemical substan‐ ces from their tank water, and their genome has been almost fully sequenced, which makes genetic manipulation more accessible. These characteristics have stimulated the use of ze‐ brafish in medicinal chemistry to assay the effects of different compounds in whole animals (Goldsmith, 2004; Kaufman & White, 2009). Another attractive characteristic of zebrafish is its potential for use in *in vivo* high-throughput screening assays. Consequently, a number of studies which take advantage of this possibility have been reported recently (Kokel et al.,

Zebrafish exhibit many social characteristics that can be assimilated to those observed in mammals. They recognize each other by sight and odor (Tebbich et al., 2002) and display an interesting social learning (Reader et al., 2003). This teleost also shows a characteristic ag‐ gressive behavior (Payne, 1998), a pheromone-mediated danger alarm (Suboski, 1988; Sub‐ oski et al., 1990), cognitive and adaptive behaviors such as habituation (Miklosi et al., 1997; Miklosi & Andrew 1999), spatial navigation abilities and Pavlovian conditioning (Hollis, 1999). These features make this species a valuable tool for either the development or the adaptation of behavioral paradigms. Thus, behavioral protocols such as an aquatic version of the T-maze, which is used for studies of discrimination, reinforcement and memory in ro‐ dents, had been used to assess color discrimination in zebrafish (Colwill et al., 2005). Anoth‐ er interesting model is the aquatic version of conditioned place preference (CPP), where the fish can be exposed to different stimuli in two separate compartments and is then allowed to freely explore the apparatus without partition (Darland & Dowling, 2001). A further para‐ digm, the novel tank diving test, has been used by different research groups (Bencan & Lev‐ in 2008; Bencan et al., 2009; Egan et al., 2009; Levin et al., 2007) as a model for anxiety. It is

2010; Kokel & Peterson, 2011; Rihel et al., 2010; Zon & Peterson, 2005).

Functional and evolutionary relationships between proteins can be reliably inferred by com‐ parison of their sequences, structures or binding sites. From a drug-discovery perspective, the study of binding site similarities (and differences) can be particularly insightful since it aids the design of selective or non-selective ligands and the detection of off-targets. In addi‐ tion, knowledge of ligand-binding site similarity could increase our understanding of diver‐ gent and convergent evolution and the origin of proteins, even in those cases where no obvious sequence or structural similarity exists. In recent years, a number of algorithms have been developed for the identification and comparison of ligand-binding sites. Even though each method has its own merits and limitations, the performance of these computa‐ tional tools is continuously improving. Advances in this field, associated with the increasing availability of structural data and reliable homology models of thousands to millions of pro‐ tein molecules, provide an unprecedented framework to investigate the mechanisms under‐ lying the molecular interactions between these proteins and their ligands, as well as to evaluate the similarities between the binding sites of related and unrelated proteins

On the basis of the foregoing, the first section of this chapter provides an overview on: a) the relevance of zebrafish as an animal model of increasing interest in pharmacology; b) the im‐ pact that MAO crystal structures and molecular simulation approaches have had on the de‐ velopment of novel MAO inhibitors, as well as comparative structural and functional information about zMAO and its mammalian counterparts; c) recent developments in com‐ putational methods to evaluate similarities between ligand-binding sites, emphasizing their usefulness for the rational design of multitarget (promiscuous) drugs.

The second part of the chapter describes unpublished results regarding a further characteri‐ zation of zMAO activity and its comparison with MAOs from mammals. Specific topics in this section include: a) the construction of homology models of zMAO, built using human MAO-A and -B crystal structures as templates; b) a three-dimensional analysis of the bind‐ ing site similarities between MAOs from different species using a statistical algorithm; c) a functional evaluation of zMAO activity in the presence of a small series of reversible and se‐ lective MAO-A and -B inhibitors.
