Drug Discovery Today Volume 11, Numbers 7/8 April 2006 REVIEWS Reviews GENE TO SCREEN Orphan enzymes could be an unexplored reservoir of new drug targets Olivier Lespinet and Bernard Labedan Institut de Ge´ne´tique et Microbiologie, CNRS UMR 8621, Universite´ Paris Sud, Baˆtiment 400, 91405 Orsay Cedex, France Despite the immense progress of genomics, and the current availability of several hundreds of thousands of amino acid sequences, >39% of well-defined enzyme activities (as represented by enzyme commission, EC, numbers) are not associated with any sequence. There is an urgent need to explore the 1525 orphan enzymes (enzymes having EC numbers without an associated sequence) to bridge the wide gap that separates knowledge of biochemical function and sequence information. Strikingly, orphan enzymes can even be found among enzymatic activities successfully used as drug targets. Here, knowledge of sequence would help to develop molecular-targeted therapies, suppressing many drug-related side-effects. Biology is exploring numerous and diverse fields, each of which discovered before, despite intensive research by thousands of is very complex and difficult to study in its entirety. For many people studying the genetics and biochemistry of Saccharomyces years, immense advances in disclosing molecular functions have cerevisiae over the past 50 years. This observation has been con- been made using reductionist approaches, such as molecular firmed repeatedly, with the cohort of genomes that have been biology. Combining genetic, biophysical and biochemical con- sequenced, at a steady pace, over the past ten years. We now know cepts and methodologies has helped to disclose details of com- that many genes have been missed by reductionist approaches plex molecular mechanisms such as DNA replication. However, and we do not have any information about the actual role of some it became clear that understanding how a living cell functions of these genes in cell metabolism, leading to the concept of requires more detail than simply adding up the operations of orphan (sometimes dubbed ORFan) genes. The OrphanMine each of the cellular constituents. Reductionist approaches are, database has been built to allow the study of taxonomically therefore, inefficient methods for fully comprehending the rela- restricted microbial genes that have no homologues in any organ- tionships between all of the components of a cell and, to go a ism (www.genomics.ceh.ac.uk/orphan_mine/orphanmine.php). step further, we need a more integrative view of the basic The most recent release of OrphanMine has compiled 44,752 mechanisms that are working together in a smooth, tuned orphan genes from 150 analyzed genomes (430,826 predicted process. Systems approaches, such as genomics, help to bring proteins). This means that, on average, 10% of the total number into place an integrative view of interactions between all genes of genes per genome are orphan genes, and this percentage still and their products. holdstruenowthatthecompletesequencesof300 genomes are This trend of using holistic approaches (rather than reduction- available in public databanks [2]. ist ones) has unearthed unexpected weaknesses in previous bio- It appears clear that the traditional approaches of biochemistry chemistry, genetics and molecular biology research. This has and genetics failed to gain access to large parts of living-cell become obvious, for example as soon as the first chromosome mechanisms. In this short review, we describe another important to be sequenced, the yeast chromosome III, was analyzed and flaw that appears because of the absence of interplay between annotated [1].Itcameasabigsurprisethatthecompletesequence biochemistry and genetic advances. We discuss another kind of contained so many open reading frames (ORFs) that had not been orphan, orphan enzymes, and underline their relevance for applied research, for example in pharmacology, medicine and Corresponding author: Labedan, B. ([email protected]) agronomy. 300 www.drugdiscoverytoday.com 1359-6446/06/$ - see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.drudis.2006.02.002 Drug Discovery Today Volume 11, Numbers 7/8 April 2006 REVIEWS Defining enzyme activities and their putative TABLE 1 sensitivity to drugs Listing orphan EC numbers in the androgen and estrogen Enzymatic processes have been analyzed in various organisms, by metabolic pathway biochemists. These studies span a wide catalogue of data that have EC number Activity been gathered over several decades – from the mere description of EC_1.1.1.63 Testosterone 17b-dehydrogenase a newly detected activity to studies that disclose many features of EC_1.1.1.64 Testosterone 17b-dehydrogenase (NADP+) an enzyme in thorough detail. As early as the 1960s, an interna- EC_1.1.1.148 Estradiol 17a-dehydrogenase tional effort was launched to organize all the pieces of information EC_1.1.1.152 3 a-Hydroxy-5 b-androstane-17-one 3 a-dehydrogenase and to check its validity and consistency. Accordingly, the Inter- national Enzyme Commission (EC) classifies enzyme activities EC_1.1.1.239 3 a (17b)-Hydroxysteroid dehydrogenase (NAD+) using EC numbers (www.chem.qmul.ac.uk/iubmb/enzyme/ EC_1.14.99.11 Estradiol 6 b-monooxygenase index.html). EC numbers comprise four digits. The first digit (from EC_1.14.99.12 4-Androstene-3,17-dione monooxygenase GENE TO SCREEN numbers 1–6) delineates the broad type of activity: (1) oxidore- EC_4.1.2.30 17a-Hydroxyprogesterone aldolase ductase, (2) transferase, (3) hydrolase, (4) lyase, (5) isomerase, and (6) ligase. The second and third digits detail the reaction that an Reviews enzyme catalyzes. For example, among the transferases (EC 2), (see Box 1). Remarkably, these orphan enzymes currently repre- glycosyltransferases form the subclass EC 2.4 and hexosyltrans- sent 39.3% of the total of the retained EC numbers – a figure close ferases form the sub-subclass EC 2.4.1. The final digit represents to one that we reported in 2005 (42.5%) – despite the description, the substrate specificity. For instance, EC 2.4.1.80 is the ceramide during this past year, of 169 new enzyme activities and the re- glucosyltransferase that catalyzes the first step of glycosphingoli- annotation of many newly sequenced genes. pid metabolism, and is the target of miglustat (1,5-(butylimino)- Interestingly, many orphan enzymes could be drug targets and 1,5-dideoxy-d-glucitol), a drug used for the treatment of the mild– identifying their sequences would be a first step for disclosing new moderate type 1 Gaucher disease in humans [3]. molecular therapies. For instance, no sequences are associated with the two testosterone 17-b-dehydrogenases (EC 1.1.1.63 and Introducing the concept of orphan enzymes EC 1.1.1.64), despite the fact that these enzymatic activities have The EC classification of enzyme activities is constantly evolving been studied intensively over the years. A deficiency in these because the curators of the Nomenclature Committee of the dehydrogenases leads to male pseudohermaphroditism with post- International Union of Biochemistry and Molecular Biology are pubertal virilization and, sometimes, gynecomastia [6]. It has been deciding on the best definition for each enzymatic activity (in proposed that these enzymes should be used as targets for the relation to their specific expertise). Note that, an EC number treatment of prostate cancer, because they are competitively denotes an enzyme activity but not the enzyme itself that catalyzes inhibited by anticancer drugs [7,8]. this activity. Thus, different sequences and structures can share the These two orphan enzymes belong to the androgen and estro- same EC number (see later). gen metabolic pathway comprising 23 EC numbers that corre- Currently (September 2005), 4499 EC numbers are indexed but spond to well-characterized enzyme activities. Interestingly, only 3877 correspond to a defined unambiguous activity. The 622 besides EC 1.1.1.63 and EC 1.1.1.64, this pathway contains six others are either in the process of deletion or have been found to be more orphan enzymes (Table 1). Considering the importance of identical to (or included with) another EC number. hormonal regulations in many aspects of human health, some of It came as a big surprise when Karp et al. [4] and Lespinet and these orphan enzymes could be potential drug targets. Indeed, Labedan [5] independently observed that a significant proportion knowing their sequences could help in designing drugs that are of EC numbers was not associated with any protein sequence. An more efficient. update of our results confirms this finding. At present, only 2352 EC numbers have at least one associated sequence in the UniProt Are orphan enzymes currently used as drug targets? Knowledgebase (Release 5.8 30th August 2005). We have assigned In a recent review, Robertson [9] listed 71 EC numbers for enzymes orphan enzymes the 1525 EC numbers found to be sequence-less that are targets for marketed drugs. These enzyme activities are in Uniprot and in public databases specializing in EC numbers present in humans (48), bacteria (13), viruses (5), fungi (4) and protists (1). Unexpectedly, we found that there were orphans even among BOX 1 enzymes used as drug targets. Three of the 71 reported activities Searching information about EC numbers in inhibited by drugs have no associated sequences. Here is the specialized databases detailed list of these orphan EC numbers used as targets for pharmaceutical drugs: Uniprot (www.expasy.uniprot.org/index.shtml) The vitamin-K-epoxide