Current Medicinal Chemistry, 2007, 14, 1291-1324 1291 Pyridoxal 5’-Phosphate as Targets for Therapeutic Agents

Alessio Amadasi#,1, Mariarita Bertoldi#,2, Roberto Contestabile#,3, Stefano Bettati1, Barbara Cellini2, Martino Luigi di Salvo3, Carla Borri-Voltattorni2, Francesco Bossa3 and Andrea Mozzarelli*,1

1Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, 2Dipartimento di Scienze Morfologico- Biomediche, Sezione di Chimica Biologica, Università di Verona, and 3Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Università di Roma La Sapienza, Italy

Abstract: The vitamin B6-derived pyridoxal 5’-phosphate (PLP) is the cofactor of enzymes catalyzing a large variety of chemical reactions mainly involved in . These enzymes have been divided in five families and fold types on the basis of evolutionary relationships and structural organization. Almost 1.5% of all in prokaryotes code for PLP-dependent enzymes, whereas the percentage is substantially lower in eukaryotes. Although about 4% of -catalyzed reactions catalogued by the Enzyme Commission are PLP-dependent, only a few enzymes are targets of approved drugs and about twenty are recognised as potential targets for drugs or herbicides. PLP-dependent enzymes for which there are already commercially available drugs are DOPA decarboxylase (involved in the Parkinson disease), GABA aminotransferase (epilepsy), hydroxymethyltransferase (tumors and malaria), decarboxylase (African sleeping sickness and, potentially, tumors), racemase (antibacterial agents), and human cytosolic branched-chain aminotransferase (pathological states associated to the GABA/glutamate equilibrium concentrations). Within each family or , the enzymes for which drugs have been already approved for clinical use are discussed first, reporting the enzyme structure, the catalytic mechanism, the mechanism of enzyme inactivation or modulation by substrate-like or transition state-like drugs, and on-going research for increasing specificity and decreasing side-effects. Then, PLP-dependent enzymes that have been recently characterized and proposed as drug targets are reported. Finally, the relevance of recent genomic analysis of PLP-dependent enzymes for the selection of drug targets is discussed.

Keywords. Vitamin B6, enzyme, drug design, pyridoxal 5’-phosphate, inhibitors, X-ray structure, functional genomics, inhibition mechanism.

INTRODUCTION reaction share several common features. The cofactor is invariantly bound through an aldimine linkage to the epsilon- Pyridoxal 5'-phosphate (PLP), one of the active forms of amino group of a Lys residue located at the enzyme active site, vitamin B6, was first identified in the mid forties as the cofactor forming the so-called "internal aldimine", while "external required for the biochemical reaction of [1]. aldimines" are referred to as those formed with the substrates Since then, PLP-dependent enzymes have been the subject of (Scheme 1b). Both types of aldimines react reversibly with extensive research. The interest aroused by these enzymes is due primary amines in a transaldimination reaction, with formation to their unequalled catalytic versatility and widespread of a geminal diamine intermediate, allowing either binding of involvement in cellular processes. Nowadays, more than 140 substrates or release of products (Scheme 1b). During , different enzyme activities based on PLP are classified by the PLP acts as an electron sink. The net negative charge arising Enzyme Commission, representing 4% of all known catalytic from the heterolytic cleavage of sigma bonds is delocalised by activities. This is because PLP-dependent enzymes are not only the extensive conjugation of the p-electrons of the pyridine involved in the synthesis, interconversion and degradation of ring, which extends to the aldimine bond and the phenolate amino acids, but also play key roles in the replenishment of substituent at the cofactor C3. The stabilised carbanion is one-carbon units, synthesis and degradation of biogenic referred to as the "quinonoid intermediate", in which the amines, synthesis of tetrapyrrolic compounds and metabolism substrate and the cofactor generate a coplanar structure (Scheme of amino-sugars. The consequence of their crucial metabolic 1b). Although most of PLP-catalysis requires the formation of a relevance is that a number of these enzymes are widely quinonoid, there are examples of concerted mechanisms that do recognised drug targets [2,3]. The outstanding catalytic not require its presence, such as in alanine racemase [8,13]. diversity of PLP-dependent enzymes arises from modulation Besides the gain in delocalisation energy, an important factor in and enhancement of the coenzyme intrinsic chemical properties the activation of sigma bonds by a p- system is their by the surrounding polypeptide matrix. The cofactor alone is stereochemical arrangement. Dunathan [14] pointed out that the indeed able to catalyze multiple reactions, though at much gain in delocalisation energy aids the bond breaking process if lower rates than enzymes [4-7]. PLP-catalyzed reactions (Scheme the transition state assumes a geometry that approaches that of 1a), which involve the heterolytic cleavage of sigma bonds at the quinonoid intermediate, i.e. the bond to be broken must be the substrate C-alpha, may be classified as reactions proceeding perpendicular to the p- system. This fundamental concept through: i) elimination of CO2 from C-alpha (alpha- explains how reaction specificity is controlled and points on decarboxylation and transaminating decarboxylation); ii) the role of the polypeptide chain in directing the coenzyme deprotonation (racemization, transamination, beta-elimination intrinsic catalytic properties. Recently, some PLP-dependent and beta-replacement, gamma-elimination and gamma- enzymes were reported to contain other cofactors, such as a replacement) and iii) elimination of the side chain of amino heme group in human beta-synthase [15,16], an acids (alpha-synthesis and aldolic cleavage) [8-12]. Despite the Fe-S cluster in mammalian GABA aminotransferase [17], and variety of catalyzed transformations, the mechanisms of adenosylmethionine in 2,3-aminomutase, biotin synthase and cystathionine beta-synthase [15,16,18,19]. This *Address correspondence to this author at the Dipartimento di Biochimica e adds a further level of complexity and regulation in PLP- Biologia Molecolare, Università di Parma, Via GP Usberti 23/A, 43100 Parma, dependent catalysis. Italy; Tel: 39-0521-905138; Fax 39-0521-905151; E-mail: [email protected] On the basis of the available structural information, it is #These authors contributed equally to this work. generally accepted that PLP-dependent enzymes originated very

0929-8673/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd. 1292 Current Medicinal Chemistry, 2007, Vol. 14, No. 12 Amadasi et al.

a

b E-Lys COO- H

+ H - NH Lys-E RH2C C COO RH2C NH NH+ - - H NH + HO3P O 2 O -HO P O- - - 3 HO3P O O O + N CH3 N+ CH + H amino acid 3 Lys N CH3 H+ H H Internal aldimine Geminal diamine External aldimine

- RH C C COO - 2 RH2C COO NH+ + + NH H NH3 - - H2C HO3P O O - - -HO P O- HO3P O 3 O O

N CH3 N CH + N CH3 3 H H H+ H+ Quinonoid Ketimine Pyridoxamine phosphate

COO-

+ -R H NH

- - HO3P O O

N CH3 H+ a-Aminoacrylate Scheme 1. Catalytic versatility of PLP-dependent enzymes (adapted from [9]) (a.). Structures of catalytic intermediates (b.). These include the internal aldimine, the geminal diamine, the external aldimine, the ketimine, the pyridoxamine 5’-phosphate and the a -aminoacrylate. For the quinonoid species, as a representative example, the intermediate formed by a -carbon removal is shown. PLP-Dependent Enzymes as a Drug Target Current Medicinal Chemistry, 2007 Vol. 14, No. 12 1293

Table 1. X-Ray Structures of PLP-dependent Enzymes

Enzyme (fold type) Ligand free, resolution (Å) Ref. Intermediate complex Ref. Inhibitor complex Ref.

g-aminobutyric acid 1OHV, 2.30 [17] 1OHW, 2.30 [17] aminotransferase (I) (vigabatrin) 1OHY, 2.80 [17] (g- ethynyl GABA) Branched chain amino acid 1KTA, 1.90 [436] 1KT8, 1.90 [436] 2A1H, 1.80 aminotransferase (IV) (ketimine with L-ile) (gabapentin) 1EKF, 1.95 [80] 2COJ, 2.40 [91] (gabapentin) 2COG, 2.10 [91] (4-methylvalerate) aminotransferase (I) 2CH1, 2.40 [109] 2CH2, 2.70 [109] (4-(2-aminophenyl)-4- oxobutyric acid) 1W7L, 2.00 [437] 1W7M, 2.70 [437] (L-) (I) 1QZ9, 1.85 [110] 3,4-Dihydroxyphenylalanine 1JS6, 2.60 [28] 1JS3, 2.25 [28] decarboxylase (I) (carbidopa) (I 1D7K, 2.10 [165] 1F3T, 2.00 [170] 2TOD, 2.00 [29] prokaryotic, III eukaryotic) () (eflornithine) 1ORD, 3.00 [163] 1NJJ, 2.45 [180] (G418) 1QU4, 2.90 [29] 7ODC, 1.60 [164] Diaminopimelate decarboxylase (III) 1KNW, 2.10 [209] 1KO0, 2.20 [209] 1TUF, 2.40 [211] (L-lysine) (azalaic acid) 1HKW, 2.80 [210] 1HKV, 2.60 [210] (L-lysine) 1TWI, 2.00 [211] (L-lysine) Alanine racemase (III) 1SFT, 1.90 [13] 1BD0, 1.60 [222] (alanine phosphonate) 1XFC, 1.90 [217] 1EPV, 2.20 [221] (D-cycloserine adduct) 1RCQ, 1.45 [218] 1NIU, 2.20 [221] (L-cycloserine adduct) 1VFH, 2.00 [233] 1VFS, 1.90 [233] (D-cycloserine) 1VFT, 2.30 [233] (L-cycloserine) 2SFP, 1.90 [223] (propionate) Serine hydroxymethyltransferase (I) 1BJ4, 2.65 [250] 1DFO, 2.40 [247] (L- and 5-formyl tetrahydrofolate) 1CJ0, 2.80 [248] 1KKP, 1.93 [249] (L-serine) 1EJI, 2.90 [246] 1KL1, 1.93 [249] (L-glycine) 1KKJ, 1.93 [249] Cystathionine g-synthase (I) 1CS1, 1.50 [277] 1I41, 3.20 [286] (APPA) 1QGN, 2.90 [278] 1I43, 3.10 [286] (PPCA) 1I48, 3.25 [286] (CTCPO) Cystathionine b-lyase (I) 1CL1, 1.83 [294] 1CL2, 2.20 [284] (L-aminoethoxyvinyl glicine) 1294 Current Medicinal Chemistry, 2007, Vol. 14, No. 12 Amadasi et al.

(Table 1). contd.....

Enzyme (fold type) Ligand free, resolution (Å) Ref. Intermediate complex Ref. Inhibitor complex Ref.

1IBJ, 2.30 [295] Cystathionine g-lyase (I) 1N8P, 2.60 [276] g-lyase (I) 1GC0, 1.70 [307] 1E5E, 2.18 (propargylglycine) 1E5F, 2.18 Cystalysin (I) 1C7N, 1.90 [27] 1C7O, 2.50 [27] (L-aminoethoxyvinyl glycine) Cystathionine b-synthase (II) 1JBQ, 2.60 [327] 1M54, 2.60 [323] O-acetylserine sulfhydrylase-A (II) 1OAS, 2.20 [337] 1D6S, 2.30 [339] (L-methionine) 1FCJ, 2.00 [338] 1Y7L, 1.55 [340] (SAT C-terminal peptide) 1Z7W, 2.20 [438] O-acetylserine sulfhydrylase-B (II) 2BHT, 2.10 [341] Glutamate 1-semialdehyde 2GSA, 2.40 [360] 3GSB, 3.00 [360] aminomutase (I) (gabaculine) 2CFB, 2.85 [362] synthase (II) 1UIM, 2.15 [373] 1V7C, 2.00 [373] (substrate analogue) 1KL7, 2.70 [374] 2C2B, 2.60 [376] (S-adenosylmethionine) 1VB3, 2.20 1E5X, 2.25 [375] 2D1F, 2.50 synthase (II) 1BKS, 2.20 [379] 1KFJ, 1.80 [392] 1A50, 2.30 [387] (L-serine) (5-fluoroindole propanol phosphate) 1KFK,2.40 [392] 1QOP, 1.80 [388] 1A5S, 2.30 [387] (indole glycerol phosphate) (5-fluoroindole propanol phosphate and L-serine) 1TTP, 2.30 [385] 1C8V, 2.20 [398] (transition state analogue) 1TTQ, 2.00 [385] 1C29, 2.30 [398] (transition state analogue) 1C9D, 2.30 [398] (transition state analogue) 1CW2, 2.00 [398] (transition state analogue) 1CX9, 2.30 [398] (transition state analogue) 1K3U, 1.70 [391] (N-(1H-indol-3-yl-acetyl) ) 1K7E, 2.30 [391] (N-[1H-indol-3-yl-acetyl.] glycine acid) 1K7F, 1.90 [391] (N-[1H-indol-3-yl-acetyl] acid) 7,8-diaminopelargonic acid 1QJ5, 1.80 [417] 1QJ3, 2.70 [417] 1MLZ, 2.15 [422] synthase (I) (7-keto-8-aminopelargonic (trans-amiclenomycin) acid) 1MLY, 1.81 [422] (cis-amiclenomycin) 8-amino-7-oxopelargonate synthase 1BS0, 1.65 [429] 1DJ9, 2.00 [432] 2G6W, 2.14 [433] (I) ((S)-8-amino-7- (trifluoroalanine adduct) oxonanonoate) 1DJE, 1.71 [429] PLP-Dependent Enzymes as a Drug Target Current Medicinal Chemistry, 2007 Vol. 14, No. 12 1295 early in the evolution (before the three biological kingdoms properties and evolutionary path, the folding mechanisms and diverged) from different protein ancestors which generated five the development of new and more efficient inhibitors, independent families, each corresponding to a different fold eventually suitable as drugs. To this latter goal, it should be type [20-22]. The families have been named from the more pointed out that structural studies on PLP-dependent enzymes representative enzyme. The aspartate aminotransferase family have provided evidence that the regulation of the enzyme corresponds to fold type I and contains the majority of activity is frequently associated with the existence of two structurally determined PLP-dependent enzymes. The conformational states: an open conformation, endowed with tryptophan synthase b- subunit family corresponds to the fold low, if any, catalytic activity, and a closed conformation, type II, the alanine racemase family corresponds to the fold type catalytically active. Interaction with the substrate or substrate III, the D-amino acid aminotransferase family corresponds to the analogues, as well as with inhibitors or allosteric effectors, fold type IV, and glycogen phosphorylase corresponds to fold affects this equilibrium and, thus, modulates the catalytic type V. Nowadays, more than a hundred crystal structures of activity. The bifunctional enzyme tryptophan synthase PLP-dependent enzymes, most of them solved in the last 5 represents a classical example [23-25]. years, are accessible from databases (Table 1). Furthermore, Because many PLP-dependent enzymes have a well thousands of sequences of PLP-enzymes have become available established involvement in human diseases and a critical role in as a follow-up of genomic projects. This wealth of information metabolic pathways in pathogens and plants, several crystal represents a frame in which the previously acquired biochemical structures were also solved in the presence of specific data may be reinterpreted and better understood and provides a inhibitors, providing insight into the mechanism of platform from which new challenges may be undertaken on the inactivation by mechanism-based inhibitors. Representative control of reaction and substrate specificity, the regulation of examples are vigabatrin bound to GABA-aminotransferase the catalytic activity, the relationship between catalytic

a. O O -O -O SO -O 3 NH3+ NH3+ NH3+ -Vinyl-GABA (Vigabatrin) g Ethanolamine-o-sulphate g-Ethynyl-GABA

H2N H2N COOH H N COOH 2 COOH cis-3-Aminocyclohex- -4-ene-1-carboxylic acid trans-3-Aminocyclohex- cis-2-Aminocyclohex- -4-ene-1-carboxylic acid -3-ene-1-carboxylic acid

H 2N COOH trans-2-Aminocyclohex- -3-ene-1-carboxylic acid

NH3+ c. PO3H- NH3+ b. +H3N SO - F 3 HOOC 2-Aminoethane phosphonic acid 2-Aminobenzene sulfonate O 4-Amino-5-fluoro-pentanoic acid

HN CH SO - F or Br Br Br 3 3

SO3- N H N COOH 2 H2N COOH H2+ H2N COOH Br F (±)Piperidine-3-sulfonic acid Rigid analogues of 4-amino-5-halo-pentanoic acid 2-(N-Acetylamino) cyclohexane sulfonic acid

CH N N 3 N H2N N C NH N C N H H H O CH3 HO Tetrazole bioisostere of vigabatrin N1-(2,6-Dimethylphenyl)-N4-(2-hydroxybenzaldehyde) semicarbazone Fig. (1). Chemical structures of GABA aminotrasferase inhibitors. 1296 Current Medicinal Chemistry, 2007, Vol. 14, No. 12 Amadasi et al.

[17,26], AVG bound to cystalysin [27], carbidopa bound to semialdehyde and enters the Kreb’s cycle. DOPA decarboxylase [28], and alpha-difluoromethylornithine Although the GABA shunt has been found to operate in bound to ornithine decarboxylase [29]. Control of reaction prokaryotes [33], mammals [34] and plants [35], it has distinct specificity is particularly important for PLP-dependent enzymes physiological functions in these . In , it plays because it is known that they catalyze side reactions at a role in carbon and metabolism [33]. In eukaryotic significant rates with substrate analogues or even with their cells, GABA-AT and succinic semialdehyde dehydrogenase are natural substrates. This ability has been proposed to play a localized in the mitochondria. In plants, GABA is more than just physiological role because it often leads to the formation of a metabolite, since its concentration level seems to be related to inactive forms of the enzyme [30]. Whereas the chemistry of oxidative stress, insect wounds and signalling [36]. In PLP-catalyzed reactions makes it easy to design effective vertebrates, GABA is the major inhibitory neurotransmitter in inhibitors, specificity remains a major challenge for their use in the central nervous system (CNS) [37]. The levels of GABA in vivo. This is the case for several inhibitors that cannot be used the CNS are determined by the relative velocities of the for pharmaceutical applications because they also inactivate reactions catalyzed by GABA-AT and other PLP-dependent enzymes. Moreover, all mechanism-based decarboxylase, the PLP-dependent enzyme which inhibitors designed for PLP-dependent enzymes bind decarboxylates glutamate, the major excitatory neurotransmitter irreversibly and the permanent modification of may in the brain. When the concentration of GABA in the brain generate new antigenic sites which could lead to an autoimmune decreases below a threshold level, convulsions result [38]. response in humans. Therefore, it is desirable that new specific However, if GABA levels are increased, the seizure comes to an and reversible inhibitors are designed. The availability of the end [39]. It is estimated that about 1% of the world population crystal structure of the target enzymes and computational is affected by recurring convulsing seizures, caused by several methods make this goal achievable. different diseases, although they are often collectively and In recent years, bioinformatic approaches have been broadly referred to as epilepsy [40]. Low GABA levels in the successfully used to discover new PLP-dependent enzymes, brain have been indeed implicated in the symptoms associated characterising those which are functionally unclassified. A with epilepsy [41,42], Parkinson’s disease [43], Huntington’s recent intra- and inter-genomic analysis of the distribution of chorea [44], Alzheimer’s disease [45] and tardive dyskinesia PLP-dependent enzymes has provided evidence that for a [46]. Therefore, it is not surprising that anticonvulsant drugs significant fraction of them activity is still uncertain or have been sought for centuries. Moreover, since about one unknown [31]. The characterisation of these enzymes may help quarter of the epileptic patients do not respond to any marketed to better define some metabolic pathways and discover new anticonvulsant medicine, the need for new anticonvulsant drugs ones, thus widening the scope of perspective therapeutic and is still urgent [40]. GABA concentration in the brain cannot be technological strategies. increased by direct oral or parental administration of the amino acid itself, since it cannot cross the blood-brain barrier. One successful strategy consists of using a compound that has AMINOTRANSFERASES access to the brain and selectively inhibits or inactivates GABA-AT, thus, increasing the concentration of GABA. A -Aminobutyric Acid Aminotransferase number of structural analogues and mechanism-based inhibitors with anticonvulsant activity have been designed and -Aminobutyric acid (GABA) aminotransferase (GABA-AT; g studied starting from the 1970s (Fig. (1a)). The first reported EC 2.6.1.19) is an ubiquitous enzyme which catalyzes the mechanism-based inhibitor was ethanolamine-O-sulphate conversion of GABA into succinic semialdehyde. The reaction [47,48], followed, some years later, by 4-amino-5-hexynoic acid mechanism is a typical reversible transamination, in which (or g-ethynyl GABA) [49]. Both the compounds never became of GABA and a -ketoglutarate are the substrates and succinic clinical interest. A compound related to g-ethynyl GABA, semialdehyde and L-glutamate are the products [32]. GABA-AT vigabatrin (4-amino-5-hexenoic acid or g-vinyl GABA), turned is one of the three enzymes which constitute the so called GABA out to be the most efficient mechanism-based inhibitor as shunt. Glutamate decarboxylase catalyzes the conversion of L- anticonvulsant agent [50,51]. Moreover, vigabatrin is effective glutamate to GABA, consuming a proton and releasing CO2.. in treating patients that are resistant to other drugs and is The succinic semialdehyde produced in the GABA-AT-catalyzed currently in use in over 60 countries [52]. transamination is converted into succinate by succinic

Fig. (2). Structures of GABA aminotransferase in the free form (a.) (PDB code 1OHV) and in complex with vigabatrin (b.) (PDB code 1OHW) [17]. PLP-Dependent Enzymes as a Drug Target Current Medicinal Chemistry, 2007 Vol. 14, No. 12 1297

The determination of the crystal structure of GABA-AT from binding of vigabatrin (Scheme 2, structure 1) to the enzyme as pig is a relatively recent achievement [53], considering the an external aldimine (structure 2). According to a proposed importance of the enzyme as a target for antiepileptic drug mechanism, an azallylic isomerisation (reaction a), therapy. The structure of GABA-AT (Fig. (2a)) is very similar to corresponding to the tautomerisation that the natural substrate that of other members of fold type I, in particular ornithine GABA normally undergoes, generates an electrophilic species aminotransferase and dialkylglycine decarboxylase. (structure 3). A nucleophilic attack of K329 (the active site Three different mechanisms for GABA-AT inhibition by lysine residue) at the terminal carbon of the vinyl group gives vigabatrin were proposed on the basis of spectroscopic and an adduct (structure 4) that easily tautomerises to structures 5 radiolabelling evidence [54], chemical intuition [51,55] and and 6. The alternative two mechanisms begin with an allylic radiochemical experiments [56]. All mechanisms start with the isomerisation through the vinyl double bond (reaction b) which

-OOC NH2 Lys 1 H N+ C H O- -2O3PO

N H

-OOC Lys

:NH H N+ 2 a C H Lys b Pyr 2 NH2: -OOC Lys -OOC NH 2 H H N+ N+ H + C H H C H 7 Pyr d Lys Pyr 3 H+ HN Lys c NH -OOC Lys -OOC -OOC + HN NH2 H CH H N+ H N H C H C H Pyr 10

Pyr 4 Pyr 8

H+ H+

Lys Lys NH -OOC HN Lys -OOC H N+ H N: -OOC 2 C H H N+ Pyr 11 H C H H N+ C H Pyr 9 Pyr 5 Lys H -OOC N Lys C H + HN H2N : Pyr

-OOC 12

H N+ C H

Pyr 6 Scheme 2. Proposed mechanisms of GABA aminotransferase inactivation by vigabatrin. 1298 Current Medicinal Chemistry, 2007, Vol. 14, No. 12 Amadasi et al. yields the electrophilic species 7. This may follow two different the blood-brain barrier. Therefore, a series of potential paths. A nucleophilic attack by K329 to the penultimate carbon vigabatrin-like inhibitors were recently designed in which the of the former vinyl bond (reaction c) leads to formation of carboxylic group was replaced with more lipophilic adducts 8 and 9. In a different path, the so called enamine bioisosteres (Fig. (1b)) [72]. One of these compounds turned mechanism, K329 attacks the cofactor imine bond (reaction d), out to have an inhibitory potency much greater than that of forming a geminal diamine (structure 10) that eliminates the vigabatrin [69], although its actual anticonvulsant action has isomerised inhibitor (structure 11). The latter attacks the not yet been tested. Some taurine analogues represent a new cofactor C4’ to give the final adduct 12. Lippert et al. [51] class of reversible GABA-AT inhibitors with Ki values close to proposed that the inactivation mechanism follows a single that of vigabatrin [73]. Since GABA-AT irreversible inhibition route, from structures 1 to 6. Metcalf [55] suggested two is hypothesized to be at the basis of its adverse effects, this possibilities in which K329 acts as a nucleophile to form either class of compounds represents a very interesting alternative to adduct 4 or 10. The enamine mechanism was ruled out by De vigabatrin treatment. Recently, aryl semicarbazones (Fig. (1c)) Biase et al. [54] on the basis of labelling and mass spectrometry have been tested as anticonvulsant agents and were shown to experiments, showing that the peptide comprising K329 bound increase GABA levels in rat models and to inhibit GABA-AT in to the radiolabelled inhibitor did not contain the cofactor. The in vitro assays [74]. same authors demonstrated that pyridoxamine phosphate was released from the inhibited enzyme, suggesting that the main Branched-Chain Amino Acid Aminotransferase final adducts were either 5 or 8. On the other hand, Nanavati and Silverman [56] showed that denaturation of the inactivated Branched-chain amino acid aminotransferase (BCAT; E.C. enzyme yielded pyridoxamine phosphate and the enamine 2.6.1.42) catalyzes the transamination of the essential branched- adduct (structure 12) in a 75:25 ratio. Crystallography and chain amino acids (, and valine; BCAAs) with polarised absorption microspectro-photometry were recently a -ketoglutarate to form the respective branched-chain a - employed to discriminate amongst the many hypothetical ketoacids and glutamate [75]. BCAT enzymes are found in both inactivation mechanisms of vigabatrin [17]. The crystal bacteria and higher organisms [76]. Mammals have a structure of GABA-AT (Fig. (2b)), solved after treatment with the mitochondrial (BCATm) and a cytosolic (BCATc) form of the inhibitor in solution, followed by crystallization, showed that enzyme [77,78], while bacteria have a single BCAT enzyme [79]. K329 in the covalent adduct is bound to the terminal carbon of Whereas most PLP-dependent aminotransferases have been the vinyl group, apparently excluding both alternative placed in the fold type I (aspartate aminotransferase family), the mechanisms which begin with an allylic isomerisation through BCATs belong to the fold type IV of the PLP-dependent the vinyl double bond (reaction b). However, the enzymes and are the only mammalian proteins in this group crystallographic analysis could not discriminate between [80]. This was revealed by the three dimensional structure of the adducts 4 to 6, and more than one of these tautomers could be human mitochondrial enzyme, that was determined in the present in the crystal. The enamine adduct reported by Nanavati ketoenamine and pyridoxamine forms [80]. Leucine, isoleucine and Silverman [56] might have been released from the enzyme and valine readily cross the blood-brain barrier, with leucine crystals over the time needed for the crystallization. Indeed, test diffusion rate higher than that of isoleucine and valine [81]. refinement of the model showed that occupancy may be set as of the BCAAs provides nitrogen for the synthesis of low as 75% without getting unreasonable B-values, glutamate and . In rat brain, BCATc accounts for about consistently with the 75:25 ratio previously reported. The 70% of all BCAT activity [82]. It has been shown that rat brain spectroscopic analysis on the crystals soaked in a vigabatrin BCAAs provide up to 30% of the nitrogen for glutamate solution provided results compatible with the mechanism synthesis in vivo [83]. The brain is unique in that it expresses proceeding through reaction a, but suggested that the two separate BCAT isoenzymes: the cytosolic form (BCATc), behaviour of the protein in solution and in the crystal is which is found in the nervous system, ovary and placenta, and different. In particular, the reaction of GABA-AT crystals with the common peripheral mitochondrial form (BCATm) which is the inhibitor would not proceed further than structure 3. found in all organs, except . The isoenzymes might play Unfortunately, vigabatrin has quite severe side effects. distinct metabolic roles. In culture systems, BCATc is Patients consuming daily 0.5 g of vigabatrin, as the requirement found only in neurons and developing oligodendrocytes, for an efficient treatment, have suffered from an irreversible loss whereas BCATm is found in astroglia and microglia [84,85]. In of their peripheral visual field [57]. The molecular mechanism the neurotransmitter system, a common function for BCATc of this defect is still unclear [58-60]. However, since visual might be to modulate the amount of glutamate available either dysfunctions are also caused by other anticonvulsant drugs, for release as neurotransmitter or for use as precursor for this side effect may be a relatively common outcome of synthesis of GABA [86]. Due to the impact of BCAAs on anticonvulsant treatment or even a feature of the natural history neuronal and astrocytic metabolism and trafficking between of epilepsy itself [61,62]. Other mechanism-based inhibitors, neurons and astrocytes, excess of BCAAs may have a profound studied in the 1970s, such as gabaculine [63,64], resulted to be influence on neurological function [87]. toxic, non-specific or poorly crossing the blood-brain barrier. The human BCAT isozymes are 58% identical in amino acid Consequently, novel GABA-AT inhibitors are urgently required sequence [88]. BCATc and BCATm show similar specificities for as therapeutic agents for epilepsy and other CNS pathologies. amino acid and a -ketoacid substrates. Both enzymes Various analogues of vigabatrin have been designed and tested transaminate BCAAs and their straight chain analogues, L- as inhibitors of GABA-AT. Some conformationally restricted alloisoleucine and glutamate. A striking difference between versions of these analogues proved to be very potent inhibitors human BCATc and BCATm is the sensitivity of the different [65,66]. A good strategy in designing mechanism-based isoenzymes to the inhibition by the neuroactive drug inhibitors of GABA is to incorporate a good leaving group in gabapentin (1-(aminomethyl)cyclohexane acetic acid) (Fig. (3)) the b-position of an amino acid that is a substrate for the [87]. Gabapentin was designed as a GABA analogue and is enzyme. 4-Amino-5-fluoro-pentanoic acid [67,68] and some of widely used for seizure control [89]. Presently, this drug is used its rigid analogues turned out to be potent inhibitors (Fig. (1b)) extensively to treat neuropathic pain, migraine headache, and [26,69-71]. Many of the inhibitors so far described contain a several other non epilectic conditions [90]. Gabapentin inhibits hydrophilic carboxylic acid group and this is undoubtedly an competitively rat and recombinant human BCATc with a Ki inconvenient feature for compounds that are supposed to cross similar to the Km for leucine, but is not an effective inhibitor of PLP-Dependent Enzymes as a Drug Target Current Medicinal Chemistry, 2007 Vol. 14, No. 12 1299 human BCATm [87,91]. The difference in this specificity of antagonist of several subtypes of glutamate receptor [101] and gabapentin is likely due to the behavior of BCATc interdomain was used as a lead compound for the development of glutamate loops and the relative orientation between the small and large antagonists that may be useful in several CNS disorders domain [91]. Because BCATc is the predominant isoenzyme [102,103]. 3-Hydroxykynurenine can generate free radicals and found in the brain and does not seem to be expressed in most contribute to, or exacerbate, neuronal damages [104]. Its levels tissues outside the human brain, gabapentin is unlikely to are elevated in cases of HIV infection, especially when influence BCAAs metabolism in peripheral tissues [92]. The associated with dementia, infantile spasm and hepatic ability of gabapentin to inhibit BCATc may be related to its encephalopathy [104]. Quinolinic acid has been increasingly antiseizure and neuroprotective activity [93]. However, implicated in mediating toxic effects in Huntington’s disease gabapentin mainly influences brain function in other ways: it [104]. Therefore, enzymes of the kynurenine pathway are binds with high affinity to the a 2d subunit of the voltage- interesting targets for rational therapeutic intervention. One of sensitive calcium channel [94] and can stimulate glutamine these, kynurenine aminotransferase (KAT; EC 2.6.1.7), is a fold efflux from astrocytes to be used for neuronal glutamate type I PLP-dependent enzyme. KAT catalyzes the transamination synthesis [87,95]. Therefore, BCATc does not likely represent of both kynurenine and 3-hydroxykynurenine to kynurenic the principal biological target of gabapentin. Other compounds acid and xanthurenic acid, respectively. Peripheral organs have been synthesized and shown to inhibit human BCATc with contain several aminotransferases capable of catalyzing these low IC50 (0.8 mM). For example, sulfonyl hydrazide compounds reactions. However, only two of these enzymes appear to be have demonstrated very high potency, selectivity and present in the brain, arbitrarily termed KAT I and KAT II [99]. favourable pharmacokinetic parameters, leading to They differ substantially in their pH optimum and substrate neuroprotection both in vitro and in vivo [96]. Indeed, because specificity, with KAT I that shows relatively low specificity, BCATc is involved in the synthesis of the excitatory while KAT II prefers L-kynurenine as substrate. To date, no neurotransmitter glutamate in the CNS, the inhibition of this preferential KAT I inhibitors have been identified. However, it enzyme has been proposed as a way to treat neurodegenerative was shown that a -aminoadipic acid, quisqualic acid and D,L-5- disorders involving disturbances of the glutamatergic system bromokynurenine are selective KAT II inhibitors in vitro [99]. [93]. These compounds may prove useful in situations in which a down-regulation of brain kynurenic acid is needed. BCAT also catalyzes, in Mycobacterium tuberculosis, Bacillus subtilis, Bacillus anthracis and Bacillus cereus, Xanthurenic acid, another product of the kynurenine methionine formation from ketomethiobutyrate [97]. The pathway (Scheme 3), plays an essential role in the regeneration of methionine consumed during polyamine gametogenesis and fertility of Plasmodium falciparum, the is an important pathway in many microorganisms. etiological agent of malaria [105]. The concentration of this In order to develop new antimycobacterial drugs, aminooxy compound required for optimal Plasmodium falciparum sexual compounds have been examined as potential Mycobacterium maturation is well above the average concentration in human tuberculosis enzyme inhibitors [98]. O-benzylhydroxylamine, plasma [106]. It follows that 3-hydroxykynurenine O-t-butylhydroxylamine, carboxymethoxylamine, and O- aminotransferase activity in infected mosquitoes could be allylhydroxylamine (Fig. (3)) yield mixed-type inhibition with essential for providing enough xanthurenic acid for sustaining Ki values in the low micromolar range. The same compounds the parasite’s gametogenesis. In fact, the 3-hydroxykynurenine were examined as antimycobacterial agents and were found to aminotransferase coding sequence turned out to be among a completely prevent cell growth [98]. These compounds group of genes which are up-regulated upon ingestion of an represent a starting point for the synthesis of BCAT inhibitors infected blood meal in distinct Anopheles species [107,108]. for treatment of tuberculosis, that remains a major world-wide The development of selective inhibitors of this PLP-enzyme health threat. may be an innovative strategy for malaria transmission- blocking drugs. The crystal structure of the enzyme from complexed with the competitive inhibitor Kynurenine Aminotransferase Anopheles gambiae, 4-(2-aminophenyl)-4-oxobutyric acid (Fig. (4)), has been In most organisms, tryptophan is primarily degraded recently reported [109]. The analysis of the enzyme-inhibitor through the kynurenine oxidative pathway that begins with the complex suggested a number of chemical modifications that opening of the indole ring and ends with the formation of NAD+ could be made on the inhibitor structure to improve its efficacy (Scheme 3). Kynurenine catabolic intermediates have received (Fig. (5)). attention for their role in the physiological tuning of the CNS and in the etiology and progression of several human Kynureninase neurodegenerative disorders [99,100]. Three of these intermediates are neuroactive: kynurenic acid, 3- Mammalian kynureninase (E.C. 3.7.1.3), another PLP- hydroxykynurenine, and quinolinic acid. Kynurenic acid, the dependent enzyme involved in the kynurenine pathway, major product of tryptophan degradation, was found to be an catalyzes the hydrolysis of 3-hydroxy-L-kynurenine to 3-

H2N COOH O O NH2 NH2

Gabapentin O-Benzylhydroxylamine O-t-Butylhydroxylamine

HOO C O O NH2 NH2

Carboxymethoxylamine O-Allylhydroxylamine Fig. (3). Chemical structures of gabapentin and other branched-chain amino acids aminotransferase inhibitors. 1300 Current Medicinal Chemistry, 2007, Vol. 14, No. 12 Amadasi et al.

NH2

COOH

N H

L-Tryptophan

O NH2 OH

COOH

NH2 Kynurenine N COOH aminotransferase Kynurenine K ynurenic acid

O NH2 OH

COOH

NH2 K ynurenine N COOH aminotransferase OH OH

3-Hydroxykynurenine Xanthurenic acid

Kynureninase L-Alanine

COOH

NH2 OH

3-Hydroxyanthranilic acid

COOH

N COOH

Quinolinic acid

NAD+ Scheme 3. Kynurenine pathway of tryptophan degradation in mammalian cells. hydroxyanthranilic acid and L-alanine (Scheme 3), whereas the Human AGXT is a liver peroxisomal enzyme that removes inducible prokaryotic kynureninase hydrolyzes L-kynurenine glyoxylate, converting it to glycine [114]. Deficiency of AGXT in and L-alanine. The structure of the enzyme leads to the conversion of glyoxylate to oxalate and the from Pseudomonas fluorescens has been solved [110] and may associated metabolic disorder hyperoxaluria type I [115]. serve as the basis for the development of selective inhibitors. Individuals suffering from this rare genetic disease face a progressive renal failure due to the accumulation of insoluble Alanine Aminotransferase calcium oxalate. AGXT also seems to be involved in the response elicited by phenelzine on patients treated for anxiety Alanine aminotransferase, also called alanine:glyoxylate disorders. Phenelzine (Fig. (6)) is a monoamine oxidase aminotransferase (AGXT; EC 2.6.1.44), is a fold type I enzyme inhibitor which is used in clinical practice to treat depression which catalyzes the reversible transamination between alanine with associated anxiety, as well as anxiety disorders such as and glyoxylate, forming pyruvate and glycine [111-113]. panic disorders and social phobia [116,117]. The PLP-Dependent Enzymes as a Drug Target Current Medicinal Chemistry, 2007 Vol. 14, No. 12 1301

[125], which is found ubiquitously in bacteria, D-glutamate is formed by two different routes of biosynthesis. In Bacillus spp. it is formed in the transamination of D-alanine and a – ketoglutarate, catalyzed by D-amino acid aminotransferase (DAA; EC 2.6.1.21) [126-128], an enzyme which is also found in higher plants [129]. On the other hand, Lactobacillus spp., Pediococcus spp. and Escherichia coli possess a glutamate racemase [130-132]. Both DAA and glutamate racemase activities were found in Staphylococcus haemolyticus [133]. DAA, just as alanine racemase, is thus an interesting target for antibacterial agents. The enzyme, which was first described by Thorne et al. in 1955 [128], is specific for D-enantiomers, but can accept as substrates various acidic, basic and neutral (aliphatic and aromatic) amino acids, which are transaminated with pyruvate, a -ketobutyrate and a -ketoglutarate [76]. The presence of an w-carboxy group, its amide or a hydrophobic unbranched chain characterises good amino donors, while the presence of a hydroxyl group, as in D-serine, D-threonine and D- Fig. (4). Structure of the active site of kynurenine aminotransferase from , prevents binding. Several typical inhibitors of PLP- Anopheles gambiae complexed with 4-(2-aminophenyl)-4-oxobutyric acid dependent enzymes inactivate DAA, although not specifically. (APOB) (PDB code 2CH2) [109]. L-Penicillamine (Fig. (6)) and D-cycloserine (Fig. (11)) are competitive inhibitors of D-alanine [76]. Potent, mechanism- neurobiological mechanisms underlying the compound efficacy based inhibitors are b–halo-D-amino acids [134] and in treating anxiety are still debated, although increasing gabaculine (Fig. (18)), although the latter inactivates several evidence suggests that GABAergic transmission may play a other PLP-dependent enzymes. major role. The effect of phenelzine is an elevation of brain levels of biogenic amines, including alanine and GABA [118- N-Acetylornithine Aminotransferase 120]. In turn, glutamine levels are decreased [119], and, since alanine is an inhibitor of glutamine synthase [121], it may be N-acetylornithine aminotransferase (AcOAT; EC 2.6.1.11) is postulated that its accumulation, conceivably due at least in a fold type I PLP-dependent enzyme catalyzing the conversion part to AGXT inhibition, may induce a secondary reduction in of N-acetylglutamic semialdehyde to N-acetylornithine in the brain glutamine levels [122]. The increase of GABA brain levels presence of a -ketoglutarate, a step involved in observed upon acute and chronic phenelzine treatments is metabolism (Scheme 4). In Escherichia coli, AcOAT also known to be caused by a sustained inhibition of another PLP- catalyzes the conversion of N-succinyl-L-2-amino-6- dependent enzyme, GABA aminotransferase [123]. An oxopimelate to N-succinyl-L,L-diaminopimelate (Dap), one of investigation that examined the effect of phenelzine on several the steps in lysine biosynthesis (Scheme 4). In vitro data amino acids levels and AGXT activity indicated a moderate but demonstrate that this enzyme exhibits both AcOAT and N- dose-related inhibition of the enzyme, which is probably succinyl-L,L-diaminopimelate aminotransferase (DapAT) contributing to the observed increase in alanine brain levels activities with very similar catalytic efficiencies and identical [124]. The analysis of the amino acid concentrations indicated kinetic mechanisms, suggesting that this enzyme can play a role an increase only for alanine and GABA, ruling out a role of in both lysine and arginine biosynthesis in vivo [135]. phenelzine as a general inhibitor of PLP-dependent The diaminopimelate/lysine biosynthetic pathway (Scheme aminotransferases. 4) provides a number of potential targets for inhibition, some of which have been shown to be essential for mycobacterial growth D-Amino Acid Aminotransferase [136]. The products of this pathway, Dap and lysine, function as the cross-linking moiety in the peptidoglycan component of The peptidoglycan layer of the bacterial cell wall is made by Gram-positive, Gram-negative, and mycobacterial cell walls. In long chains, cross-linked by peptide bridges addition, lysine is an in mammals, which contain D-amino acids, such as D-alanine and D- suggesting that inhibition of this pathway would be glutamate. While D-alanine is synthesized by alanine racemase

O O NH2 O NH O 2 Br NH3+ HN N COO- COO- -OOC COO- COO- NH O 2 NH2 a -Aminoadipic acid Q uisqualic acid DL-5-Bromokynurenine 4-(2-Aminophenyl)-4-oxobutyric acid

Fig. (5). Chemical structures of kynurenine aminotransferase inhibitors.

OH H H COOH N N C +H3N C C NH2 HS O N O O SO CH H NH2 2 3 Phenelzine L-Penicillamine Pantocin B Fig. (6). Chemical structures of PLP-dependent enzyme inhibitors. 1302 Current Medicinal Chemistry, 2007, Vol. 14, No. 12 Amadasi et al. bactericidal but without effect on lysine metabolism in relationship studies carried out on synthetic derivatives of mammals. Since AcOAT/DapAT functions in two critical amino pantocin B have provided indications on the role of the various acid biosynthesis pathways, it is a strong candidate for parts of the molecule in conveying antibiotic activity [140]. inhibitor design. AcOAT was shown to be inhibited in a The N-terminal fragment, essential for cellular import but not competitive way by pantocin B (Fig. (6)) [137], an antibiotic for interacting with the intracellular target, AcOAT, tolerates a produced by the bacterium Erwinia herbicola. E. herbicola has wide variety of substitutions as long as the key stereocenter is been found to produce an array of antibiotics that suppress the preserved. The methylenediamine and substituted succinic acid growth of the closely related bacterium E. amylovora, the fragments likely interact extensively with the target, and little, pathogen responsible for the plant disease fire blight, a if any, structural variation is tolerated. A full explanation of devastating disease of pome fruit plants, most notably apple these results will have to wait for the determination of the three- and pear trees. E. herbicola (syn. Pantoea agglomerans) dimensional structure of AcOAT. colonizes the same plant surfaces as E. amylovora, but is not pathogenic to the plant [138]. The antibiotics produced by E. DECARBOXYLASES herbicola have different patterns of biological activity and, presumably, chemical structures. Pantocin B contains unusual 3,4-Dihydroxyphenylalanine (DOPA) Decarboxylase structural elements, most notably the methylenediamine moiety, which has been reported in the structure of only one DOPA decarboxylase (DDC; EC 4.1.1.28) is a homodimeric other antibiotic, and a succinic acid fragment that is also not stereospecific a -decarboxylase belonging to the fold type I of common in natural products [139]. Structure-activity PLP-dependent enzymes. The enzyme is found in neural and

NH + 3 NH3+ O O- -OOC -OOC O- O L-Aspartate L-Glutamate

NH-Ac NH-Succ O H -OOC -OOC COO- O N-Acetyl-L-glutamate semialdehyde N-Succinyl-L-2-amino-6-oxopimelate

N-Acetylornithine N-Succinyl-L,L-diaminopimelate aminotransferase aminotransferase

NH-Ac NH-Succ NH3+

NH3 + -OOC -OOC COO-

N-Acetyl-L-ornithine N-Succinyl-L,L-diaminopimelate

NH3+ NH3+

NH3+ -OOC COO- H N NH2 meso-Diaminopimelate -OOC

NH2+ Diaminopimelate L-Arginine decarboxylase NH3+

-OOC NH3+

L-Lysine

Scheme 4. Reactions catalyzed by N-acetylornithine aminotransferase/N-succinyl-L,L-diaminopimelate aminotransferase and diaminopimelate decarboxylase. PLP-Dependent Enzymes as a Drug Target Current Medicinal Chemistry, 2007 Vol. 14, No. 12 1303

H RH2C R' Lys303-E RH2C R' NH+ NH+ NH+ -HO P -HO P 3 O- 3 O- -HO3P O- O O O

N+ CH3 N+ CH3 N CH3 + H H H H

Internal aldimine 4'-N-Protonated external aldimine Quinonoid O2 H N OH R= or

OH RH2C R' Lys303-E NH+ NH+ H C OOH -HO P -HO3P O- 3 O- RH2C R' O O NH3 + +

O N+ CH3 N+ CH3 H H Hydroperoxy-PLP

Scheme 5. Proposed mechanism for the oxidative deamination of DOPA decarboxylase. peripheral tissues, notably liver and kidney. Naturally involvement of DDC in the biosynthesis of these occurring and recombinant enzymes from either pig kidney neurotransmitters, the enzyme has been implicated in a number [141] or rat liver [142] have been purified and characterized. The of clinical disorders, including Parkinson’s disease and main catalytic activity of DDC is the decarboxylation of L- hypertension. aromatic amino acids into their corresponding amines. The Like other PLP-enzymes, DDC catalyzes side reactions with physiologically recognized substrates of the enzyme are L- turnover numbers of the order of minutes. In particular, DDC DOPA and L-5-hydroxytryptophan (L-5-HTP), but, at least in exhibits a half- activity toward D-aromatic amino vitro, the enzyme exhibits a much broader substrate specificity. acids accompanied by a cyclic condensation between D- For this reason, the enzyme is more correctly named as L- aromatic amino acids and bound PLP [143], and an oxidative decarboxylase. The decarboxylation of L- deamination toward aromatic amines [144,145] and the D- DOPA and L-5-HTP results in the formation of dopamine and enantiomer of tryptophan methyl [146]. The oxidative serotonin, respectively, which are two of the major deamination reaction is a striking feature of the enzyme in that neurotransmitters of the mammalian nervous system. Due to the DDC lacks any of the prosthetic groups or cofactors normally

O- ArH C O Lys303-E 2 ArH2C C CH2 NH+ NH-Lys303 O -HO P 3 O- -HO3P O- ArH2C CH2 O O H N+ CH 3 N+ CH3 B: H H 3,4-Dihydroxyphenylacetone Internal aldimine

ArH2C O H2N Lys-E

OH HO -HO3P O- Ar = O

N+ CH3 H Scheme 6. Proposed mechanism of DOPA decarboxylase inactivation by a -methylDOPA. 1304 Current Medicinal Chemistry, 2007, Vol. 14, No. 12 Amadasi et al. associated with oxygen chemistry (Scheme 5). Although a Parkinson’s disease involves a progressive loss of quinonoid intermediate of this novel PLP-dependent reaction dopamine-producing cells in the midbrain, and L-DOPA, given (in addition to the external aldimine) has been detected [146], orally or intraduodenally, remains the most effective treatment the complete elucidation of the mechanism of oxidative for this disorder, reducing symptoms as bradykinesia or deamination reaction may be useful in the design of rigidity. However, since L-DOPA is rapidly converted to mechanism-based inhibitors. A similar oxidative deamination dopamine in the bloodstream, it is routinely administered with reaction has been demonstrated to be operative in other a - a DDC inhibitor (carbidopa or benserazide), so that greater decarboxylases, such as Escherichia coli glutamate amounts of L-DOPA can reach the brain. Both carbidopa and decarboxylase and Lactobacillus 30a ornithine decarboxylase benserazide do not cross the brain-blood barrier. In toward their a -methyl substrates [30]. combination with L-DOPA (Sinemet®(L-dopa/carbidopa) and a -MethylDOPA, a compound considered to be a centrally Madopar®(L-Dopa/benserazide)), they increase the amount of L- DOPA at the CNS, extending its plasma half-life from 50 to 90 acting antihypertensive agent, in vitro behaves as a substrate and as an inhibitor of DDC. DDC catalyzes the cleavage of a - min. This effect allows the reduction of the drug dose and side methylDOPA into 3,4-dihydroxyphenylacetone and , effects due to the presence of dopamine, outside of the CNS. Pharmacokinetic and metabolic studies of benserazide in via the intermediate a -methyldopamine, which does not accumulate during catalysis [145]. It has also been animals and man have shown that this compound is demonstrated that the mechanism of a -methylDOPA-induced metabolized before it reaches the arterial blood, forming serine inactivation results from the covalent linkage of 3,4- and 2,3,4-trihydroxybenzylhydrazine (Ro 4-5127) [157]. This dihydroxyphenylacetone to PLP, with C3 of the ketone attached finding strongly suggested that Ro 4-5127 represents the actual to the aldehydic carbon of the coenzyme in an aldol type DDC inhibitor. The interaction of DDC with carbidopa, adduct, thus trapping the PLP-lysine aldimine of the active site benserazide and Ro 4-5127 was investigated. While benserazide behaves as a poor inhibitor (K ~ 0.3 mM) [158], either (Scheme 6). Therefore, 3,4-dihydroxyphenylacetone behaves as i an active site directed affinity label with an inactivation carbidopa [159] or Ro 4-5127 [160] bind to the enzyme by a hydrazone linkage with the PLP cofactor through its hydrazine efficiency, k /K , equal to 364 M-1min -1. inact i moiety, and have been found to be powerful irreversible Studies demonstrated that a group of DDC inhibitors is inhibitors. However, because hydrazine derivatives can react represented by a -chloromethyl and a -fluoromethyl derivatives with both free PLP and all PLP-enzymes, these inhibitors are not of DOPA [147-151], a -vinyldopa and a -acetylenic DOPA [152], selective for DDC. which have been shown to behave as highly potent irreversible The atomic structures of ligand-free DDC and DDC- and selective inhibitors of DDC. They act via an enzyme- activated “suicide mechanism”. Inactivation occurs because carbidopa complex were solved [28] (Fig. (8)), revealing several DDC decarboxylates these compounds, generating a highly features of the enzyme: the overall structure of the protein, the reactive intermediate that alkylates the enzyme. Schiff base were mode of anchoring the coenzyme to the enzyme, the mode analogs (phosphopyridoxyl aromatic amino acids) [153,154] of binding of the antiparkinsonian drug, and which amino acid and substrate analogs (catechol- or indole-related structures) residues might be involved in the catalytic activity. As in all [155,156] have been shown to bind to the active site of DDC enzymes of the fold type I, DDC consists of a large domain, and/or to inhibit decarboxylase activity. Their dissociation or containing the PLP binding site, and a C-terminal small domain. inhibition constants range from 10-2 to 10-6 M , depending on A peculiar feature of DDC is that an additional N-terminal the chemical structure of the analog. Among the substrate domain of one monomer packs on the top of the other monomer, analogs endowed with a substituted hydrazine function, L- resulting in an extended dimer interface. The orientation of the a -methyl-a -hydrazino-3,4-2,3,4-trihydroxyphenylpropionic inhibitor at the active site is well defined, with its carboxylate acid (carbidopa (MK 485)), or N-(DL-seryl) N’-2,3,4- moiety approximately orthogonal to the PLP ring and its 3’ and trihydroxybenzyl-hydrazine (benserazide (Ro-4-4602)), deserve 4’ catechol hydroxyl groups hydrogen bonded to the hydroxyl attention because they are compounds used clinically, in groups of the phosphate of the coenzyme and Thr82, combination with L-DOPA, to treat Parkinson’s disease (Fig. respectively. Furthermore, a short stretch of amino acids (residues 328-339) was not detected in the electron density map (7)). both in the ligand-free and in the carbidopa-DDC structures. HO OH These residues belong to a mobile loop that seems to be HO important for catalysis. Among these residues, Tyr332 has been CH3 HO HO COOH NH NH NH C O +H3N

NH2 HO Carbidopa Benserazide

HO OH

HO

NH

Ro 4-5127 NH2 Fig. (8). Structure of the active site of DOPA decarboxylase complexed Fig. (7). Chemical structures of inhibitors of DOPA decarboxylase. with carbidopa (PDB code 1JS3) [28]. PLP-Dependent Enzymes as a Drug Target Current Medicinal Chemistry, 2007 Vol. 14, No. 12 1305 identified as an essential residue for amine formation [161]. A addition, DFMO undergoes decarboxylation and forms a binding model for benserazide, proposed on the basis of the covalent bond with Cys360, as previously proposed for mouse DDC-carbidopa complex, has allowed the identification of an ODC (Fig. (10)). DFMO is an approved drug for the treatment of additional hydrogen bond interaction between the coenzyme African sleeping sickness caused by parasitic Trypanosoma phosphate group and a hydroxyl substituent of the aromatic brucei. It is also active in the treatment of Pneumocystis ring [28]. These findings represent relevant information for the pneumonia in AIDS patients, and may have therapeutic design of more efficient and selective inhibitors of DDC. potential for a number of other illnesses caused by protozoa and for diseases involving abnormal cellular proliferation, including cancer [171,172]. The trade name of eflornithine as Ornithine Decarboxylase manufactured for the treatment of sleeping sickness is Ornithine decarboxylase (ODC; E.C. 4.1.1.17) is a PLP- Ornidyl®. Considering that the ODC active site is invariant dependent enzyme catalyzing the initial and rate-limiting step between the host and parasite, a selective toxicity of DFMO for in the biosynthesis of polyamines, the decarboxylation of L- the parasite cannot be due to a differential inhibitor binding to ornithine (L-Orn) to form the diamine putrescine. Polyamines the enzyme active site [29], but rather to metabolic differences are not only essential for normal cell growth and differentiation between the host and parasite. They mainly consist in a more but also play an important role in cell proliferation and tumor rapid turnover of the host ODC when compared to that of the development. Eukaryotic ODCs are, in general, highly specific parasite [173,174], and in the requirement for the polyamine for L-Orn with a weak activity on L-lysine and even lower on L- spermidine of the cofactor trypanothione in order to maintain arginine [162]. The X-ray structures of native enzymes from reduced thiol pools in the parasite [175]. The discovery that Lactobacillus 30a [163], mouse [164], Trypanosoma brucei DFMO was curative against Trypanosoma brucei gambiense [29], and human [165] have been solved. The prokaryotic infections provided the first alternative to highly toxic enzyme has the same fold (fold type I) of aspartate melarsoprol for the treatment of late stage disease [176]. aminotransferase [163]. There are two domains in each However, utilization of DFMO is hampered by the large dose monomer, an N-terminal domain forming a a /b barrel and requirement and by the poor activity against the Trypanosoma containing the PLP-binding lysine, and a C-terminal domain brucei rodiense strain of the parasite [169]. A number of which has predominantly a b-sheet structure. The active sites are inhibitors (substrate or product analogues) of ODC have been formed at the dimer interface between the N-terminal domain of reported, but they target the highly conserved active site [177- one subunit and the C-terminal domain of the other. An unusual 179]. To overcome the poor pharmacology of DFMO, a strategy property of ODC is that the association between two subunits is consisting in the identification of non-substrate-based quite weak, and the dimers are in rapid equilibrium with inhibitors has been undertaken. By computational methods inactive monomers, under physiological conditions. The Jackson et al. [180] have identified as potential inhibitor of structure of eukaryotic ODC reveals that the enzyme is ODC, G418 sulfate (Geneticin, Fig. (9)). G418 behaves as a weak structurally homologous to the bacterial and plant arginine non-competitive inhibitor of ODC with respect to L-Orn (Ki ~ 8 decarboxylases, bacterial diaminopimelic acid decarboxylase mM). Structural analysis of Trypanosoma brucei ODC bound to and alanine racemase (fold type III), but unrelated to the D-Orn and to G418 (ODC/G418/D-Orn) reveals that the G418 bacterial ODCs. binding site is at the junction between the b-sheet and the a /b- barrel domain. This is consistent with the non-competitive a -Difluoromethylornitine (DFMO, Fig. (9)), also known as kinetics observed for the inhibition of ODC by G418. A eflornithine, was designed as an enzyme-activated irreversible significant order-to-disorder transition of the loop region inhibitor of ODC [166]. Indeed, incubation of the eukaryotic (residues 392-401), located at the dimer interface and enzyme with DFMO leads to the irreversible loss of activity. The containing important residues for enzyme activity, is observed mechanism of action of DFMO has been investigated [167]. The in the ODC/G418/D-Orn structure when compared with other major inactivating adduct formed between DFMO and ODC is ODC structures. The disordering of this region has been generated by a nucleophilic attack of Cys360 on the conjugated interpreted as the structural basis to explain the inhibition of imine (Scheme 7). Cys360 is located at the active site and plays ODC by G418. This finding could be useful in the design and an essential role in ensuring correct protonation of the development of allosteric inhibitors of ODC. In this regard, it is decarboxylated reaction intermediate at Ca . If Cys360 is of interest that the antizyme, a regulatory protein synthesized in mutated to Ser or Ala, there is a large reduction in activity [168], mammalian cells in response to high polyamine levels, binds at and the mutated enzyme becomes a decarboxylation-dependent a site distant from the active site, inhibiting ODC activity and transaminase due to the frequent protonation at C4’ of the targeting the enzyme for degradation [181]. intermediate [169]. The Trypanosoma brucei structure has been solved in complex with the product putrescine and with DFMO [29,164,170], identifying important active site residues. Both Decarboxylase putrescine and DFMO form a covalent Schiff base complex with the cofactor. The side chain amino groups of both compounds Histidine decarboxylase (HDC; EC 4.1.1.22) is the PLP- bind in a well conserved pocket that bridges the subunits. In dependent enzyme responsible for the catalytic production of histamine from L-histidine. Histamine plays important roles in

CH3 HO F O F +H N COOH HO 3 O CH +H N NH3+ 3 3 NH + HO 3 NH + NH3+ 2 O HO OH O CH3 a-D ifluoromethylornithine G418 (Geneticin) (eflornithine)

Fig. (9). Chemical structures of ornithine decarboxylase inhibitors. 1306 Current Medicinal Chemistry, 2007, Vol. 14, No. 12 Amadasi et al.

-S Cys380-E O O- CHF CHF F +H3N C C +H3N NH+ NH + CO2

-HO3P O- -HO 3P O- O O

N+ CH3 N+ CH3 - H F H

External aldimine

Cys360-E Cys360-E S S CH C F +H3N +H3N N NH+

-HO3P -HO3P O- O- O O

N CH3 N+ CH3 H H F-

E-Lys NH2

Internal aldimine

S Cys360-E Cys360-E S N +H3N

NH2

NH3 Scheme 7. Proposed mechanism of ornithine decarboxylase inactivation by a -difluoromethylornithine. a number of physiological processes. In addition to its function analogues, such as the inhibitors a -fluoromethylhistidine and in immune and inflammation responses [182,183], histamine is histidine methyl ester [206]. Nevertheless, it has recently been considered to be a modulator of gastric acid secretion in the reported that the natural polyphenol epigallo-catechin-3-gallate stomach [184,185], a neurotransmitter involved in memory, acts as an inhibitor of HDC [207]. appetite and circadian rhythms regulation [186-188], and a modulator of cell growth [189-191]. Histamine is also essential in angiogenesis, cellular differentiation of mast cells, and bone loss osteoporosis [186-192]. Therefore, HDC is a potential target for therapeutic intervention in many inflammatory diseases, some neurological and neuroendocrine disorders, osteoporosis and even several types of neoplasias [193-196]. The mature, catalytically active, HDC is formed by C-terminal processing of the » 74 kDa primary product which, during tissue-specific post-translational modification, generates multiple truncated isoforms [197-201]. The intracellular location and the characterization of these isoforms need to be taken into account for any inhibition strategy. Due largely to the enzyme instability, the crystal structure of HDC has not yet been determined. This has prevented the development of efficient HDC inhibitors that can be used pharmacologically. Up to now, the suicide inhibitor a - fluoromethylhistidine has been useful for basic research on Fig. (10). Structure of the active site of ornithine decarboxylase from histamine physiology and molecular biology [202-205]. HDC Trypanosoma brucei complexed with a -difluoromethylornithine (DFMO) is only able to bind histidine or imidazole-containing (PDB code 2TOD) [29]. PLP-Dependent Enzymes as a Drug Target Current Medicinal Chemistry, 2007 Vol. 14, No. 12 1307

During the last few years, by comparison with the crystal analogy, inhibition of an auxiliary product, such as structure of the homolog enzyme DDC, a three-dimensional DapDC, could resensitize resistant strains of S. aureus to beta- model of a rat HDC dimer has been built [208], and a number of lactam antibiotics. residues and motifs, important for HDC catalysis and shared by mammalian DDC, have been identified [208]. OTHER PLP-DEPENDENT ENZYME DRUG TARGETS Diaminopimelate Decarboxylase Alanine racemase Bacteria have evolved strategies for the synthesis of lysine Alanine racemase (AlaR; EC 5.1.1.1) catalyzes the from aspartate via formation of the intermediate interconversion between L- and D-alanine. The enzyme is a diaminopimelate, a metabolite that is also involved in homodimer belonging to the fold type III of PLP-dependent peptidoglycan formation. Diaminopimelate decarboxylase enzymes [8,13]. AlaR is universal to bacteria, including (DapDC; E.C. 4.1.1.20) catalyzes the final step of lysine mycobacteria, and, with a few exceptions [213-216], is absent in biosynthesis converting meso-diaminopimelate to L-lysine eukaryotes. This taxonomical restriction and the absolute (Scheme 4). Unlike bacteria, humans obtain L-lysine from requirement for D-alanine in biosynthesis of prokaryotic cell dietary sources. Therefore, DapDC represents a potential target walls, make AlaR an attractive target for inhibitors that might for broad-spectrum inhibitors, either for use as primary function as antibiotics [217,218]. Actually, D-alanine is one of antibiotics or as adjuncts to existing therapies. DapDC is the central molecules of the cross-linking step of the assembly structurally very similar to eukaryotic ODCs [164,165,169] and, of peptidoglycan, which is the backbone of bacterial cell wall. with the exception of a rotation of the C-terminal domain, to The cell wall is an effective barrier that contributes to drug Bacillus stearothermophilus alanine racemase [13]. The resistance, therefore the inhibition of its biosynthetic pathway structure of DapDC has been solved from various sources, such can also increase the susceptibility of bacteria and mycobacteria as Escherichia coli, Mycobacterium tuberculosis and to other therapeutical agents, with a synergistic effect [219]. In Methanococcus jannaschii, both in its ligand-free form particular, AlaR has been proposed as a promising target against [209,210], and complexed with the product lysine [209-211] or tuberculosis [220]. Although tuberculosis mainly affects the with the substrate analog azalaic acid [211]. Third World, it is still a major concern in developed countries, Several competitive inhibitors of DapDC, including azalaic mainly due to infection involving patients with AIDS and after the advent of multidrug-resistant Mycobacterium tuberculosis acid (Ki=89±15 mM) and the dead-end product lysine (Ki>1 mM), were identified and characterized. Dicarboxylic acids bind [217]. with low affinity to DapDC, while diamines appear to be much A number of effective inhibitors of AlaR have been reported stronger inhibitors. However, their strong inhibitory effect is and most are structural analogues of D-Ala [219,221-229]. The due to PLP sequestration which precludes recycling of the wide majority of these compounds are mechanism-based enzyme after inhibitor binding [211]. Although azelaic acid and inhibitors that react with PLP, inhibiting the activity of many diamines bind DapDC with good affinities, none of these highly PLP-dependent enzymes and, consequently, showing lack of polar molecules (with predicted poor membrane permeability) specificity and safety. Fluoroalanine and analogous b- inactivate DapDC in vivo [211]. Thus, it was suggested that a - substituted are representative examples (Fig. (11)) fluoromethyl analogs of diaminopimelate may represent lead [228,229]. D-cycloserine (D-4-aminoisoxazolidin-3-one, DCS) compounds for the development of DapDC inhibitors because is the only inhibitor of AlaR that has been marketed and is the fluorine moiety should increase membrane permeability clinically used (Fig. (11)). It is a cyclic structural analogue of D- [212]. As for the Mycobacterium DapDC, the comparison of the Ala and is produced by Streptomyces garyphalus and structure of the enzyme with the inhibitor and product-bound Streptomyces lavendulae [230]. The cycloserine inactivation of ODC structures [164,170] of the parasitic flagellate AlaR has only recently been elucidated (Scheme 8) [221,231]. Trypanosoma brucei indicates that DapDC is essential for M. DCS acts as a suicide inhibitor of AlaR. Its mechanism is tuberculosis viability and could be a potential anti- characterized by an initial transamination step, followed by mycobacterial drug target. Although there are currently no tautomerization to form a stable aromatic product (Fig. (12)). known drugs that target DapDC, one of the most widely This proposed mechanism has been supported by the structure employed drugs used to treat African sleeping sickness is of AlaR from Bacillus stearothermophilus, inhibited by L- and DFMO (Fig. (9)), a suicide inhibitor that targets T. brucei ODC D-cycloserine [221,232]. DCS is mainly employed as a second [170]. An energy-minimized model, starting from a line anti-tuberculosis agent, but its clinical use is restricted diaminopimelate molecule placed as the bound DFMO in the T. because of serious side effects, such as CNS toxicity [217]. brucei X-ray structure, shows that the same conformation is O conceivable for a putative DapDC-inhibitor complex. N CH3 CH2F Stereospecificity of the decarboxylation reaction preceding the 2- -OOC O3P attack of the reactive imine intermediate would likely HO H NH2 NH2 require that a diaminopimelate analog be stereospecifically H2N H H fluorinated at the D-aminocarboxyl group of diaminopimelate [210]. D-Cycloserine (R)-1-Aminoethylphosphonic acid b-Fluoro alanine It is known that inhibition of an product Chemical structures of D-cycloserine and other alanine often fails to kill a pathogenic bacterium because the Fig. (11). in vivo racemase inhibitors. host milieu supplements the missing reaction product(s). Although this is extremely unlikely for DapDC, given the high In the perspective of developing more specific inhibitors for concentrations of L-lysine required to overcome loss of enzyme AlaR via a structure-based drug design, crystallographic activity in vitro, this scenario remains a formal possibility. structures of AlaR from four species (Bacillus Even in this context, an inhibitor of DapDC may be of clinical stearothermophilus, Pseudomonas aeruginosa, M. tuberculosis utility in combination with a beta-lactam antibiotic. In the and S. lavendulae) have been solved. The structures of all four widely used combination drug Augmentin®, the inclusion of enzymes are very similar and a detailed comparison has been clavulanic acid inhibits beta-lactamase, thereby allowing the reported [217]. Furthermore, structural evidence suggests that antimicrobial component amoxycillin to reach its target. By the larger volume and the more rigid conformation of the 1308 Current Medicinal Chemistry, 2007, Vol. 14, No. 12 Amadasi et al.

HO N O

H2N H Lys39 D-Cycloserine

+HN

-O 2- OPO3

H C N 3 H+

N O Fig. (12). Structure of the active site of alanine racemase from Bacillus stearothermophilus complexed with D-cycloserine (DCS) (PDB code HO 1EPV) [221]. H H N-Lys39 +HN 2 residues and one tyrosine have been found to be involved in hydrogen bonding with both the PLP and inhibitors or substrate analogues [13,218,221,222,234], including the -O 2- OPO3 competitive inhibitor propionate [223]. Structures of the enzyme with the inhibitors alanine phosphonate and DCS demonstrated that inhibition of AlaR often occurs the H3C N via H+ formation of a stable covalent bond with PLP [221,222]. Achieving specificity and less toxicity to humans, via a non- covalent linkage to AlaR, would be of great interest. Several approaches have been proposed after inspection of AlaR three- dimensional structure [218]. The first straight-forward method is to design an inhibitor that can bind within the enzyme active N O site, which contains several excellent candidates for hydrogen bonding as well as hydrophobic interactions [218]. This HO H :Base strategy has already been pursued, also applying computational methods that better exploited the available structural +HN information. Electrostatic potentials were calculated and ionization states of active site residues were predicted [234]. -O Tyr265, within the active site, might have an unusually low pK OPO 2- a 3 of 7.9 and, at pH 7.0, an average charge of –0.37. It follows that 37% of the Tyr265 residues, in an ensamble of enzyme H C N 3 H+ molecules, should be in the phenolate form. Furthermore, the authors stated that this phenolate side chain, that is in close proximity to the a -hydrogen atom of the substrate, appears to be a unique feature of AlaR, proposing a strong coupling to the phenolate ion as a useful feature for selective binding. Mustata and Briggs developed a receptor-based pharmacophore model N O for AlaR [235]. Different protein conformations were obtained using molecular dynamics simulations in order to take into HO account protein flexibility, and a dynamic pharmocophore model was built. Compounds fitting the pharmacophore model +H2N were identified from the Available Chemicals Directory. Other molecular dynamics studies identified a conserved water -O molecule, within the binding site, bridging substrate and OPO 2- 3 inhibitors with protein residues [236]. This water molecule might be taken into account for the design of specific AlaR H C N 3 H+ inhibitors, by either utilizing it as a bridging group or displacing it with an entropic gain, in keeping with the results Scheme 8. Proposed mechanism of alanine racemase inactivation by D- of an analysis of the interaction of water molecules within the cycloserine. free and ligand-bound enzyme active sites [237,238], using Streptomyces AlaR active site confer to the microorganism HINT as a code for the evaluation of the binding free energy resistance to its own product DCS [233]. Structural studies [239-241]. However, the size of the AlaR binding pocket and, established that each monomer of the homodimeric enzyme mostly, the wide size of the active site entrance cast some consists of two different domains, an a /b-barrel at the N- doubts on the possibility of designing specific AlaR inhibitors terminus and a C-terminal domain with a novel architecture [218]. Recent studies have shown that the regions of the composed primarily of b-strands. The PLP cofactor is located corridor constituting the entryway to AlaR active site are within the core of the a /b-barrel, where it forms an internal strongly conserved across species [217]. Finally, the inhibition aldimine linkage with a lysine residue [217]. Two arginine of the enzyme dimerization could be another way to inactivate PLP-Dependent Enzymes as a Drug Target Current Medicinal Chemistry, 2007 Vol. 14, No. 12 1309 the enzyme [240]. This approach has not yet been used for AlaR, dihydrofolate reductase and have been used for anticancer but has been proposed for other enzymatic targets, as HIV-1 therapy for more than 50 years. 5-Fluorouracil and Tumodex are protease [241,242]. Recently, a cluster analysis of water clinically established inhibitors of thymidylate synthase. molecules and a 2 ns molecular dynamics simulation were Inhibitors of these enzymes have been also used as antibacterial carried out to gain insight into the stabilization of the dimer and antiprotozoal agents [260-262]. However, the emergence of interface [243]. The data indicated that water molecules located drug resistance is a common problem [263], hence the need to at the interface between the two monomers may play a structural identify additional targets for cancer therapy, such as SHMT, is role in the stabilization of AlaR dimer. On the other hand, felt. whether AlaR is always dimeric is still a matter of debate [240]. The only antifolate compounds with anticancer activity found to inhibit, apparently irreversibly, SHMT were sulphonyl Serine Hydroxymethyltransferase fluoride triazine derivatives [264] (Fig. (13)). The search for selective serine analogues as inhibitors has been much less Serine hydroxymethyltransferase (SHMT; EC 2.1.2.1) is successful. DCS (Fig. (11)) was found to inactivate selectively found in all prokaryotes and eukaryotes [244]. In eukaryotic SHMT in mouse liver extracts and in the liver of mice receiving organisms, the enzyme is expressed as cytosolic and a diet deficient in PLP [265]. However, it has long been known mitochondrial isoforms. Its major physiological role is to that this compound also inactivates other PLP-dependent catalyze the reversible transfer of the Cb of serine to enzymes, such as alanine aminotransferase and aspartate tetrahydropteroylglutamate (H4PteGlu) to form glycine and aminotransferase [76]. 4-Chloro-L-threonine (Fig. (13)) was 5,10-methylene-H4PteGlu. In the absence of H4PteGlu, SHMT shown to be a mechanism-based inactivator of SHMT [266] and also catalyzes at appreciable rates the aldol cleavage of several thiosemicarbazide turned out to be a slow but tight binding L-3-hydroxyamino acids and the racemization and inhibitor. However, the dubious selectivity of these compounds transamination of both L- and D-alanine. The catalytic has never been tested. mechanism of the mammalian and the Escherichia coli enzymes has been thoroughly investigated [245]. The crystallographic Mimosine (Fig. (13)), a naturally occurring plant amino acid structure of SHMT from five different sources has been solved which is an effective cell-specific inhibitor of DNA replication in the absence and presence of ligands [246-250]. SHMT plays a in mammalian cells and approved as an anti-neoplastic drug, central role in one-carbon metabolism since 5,10-methylene- has been proposed to act through the inactivation of SHMT. Mimosine was shown to bind to SHMT in crude Chinese H4PteGlu is the major one-carbon donor in many methylation reactions, in the biosynthesis of methionine, and formyl hamster ovary cells [267], whereas it does not inactivate the enzyme in vitro. Iron deficiency and iron chelators, which can tRNA [251]. Notably, 5,10-methylene-H4PteGlu participates directly in pyrimidine biosynthesis by donating a methyl chelate zinc, are known to alter folate metabolism in mammals. group to 2’-deoxyuridine monophosphate, forming 2’- It has been shown that mimosine, which is also a strong iron deoxythymidine monophosphate, and, indirectly, in purine chelator, attenuates SHMT by chelating zinc. This causes the disruption of zinc finger transcription factors with biosynthesis via its conversion to 10-formyl-H4PteGlu. Glycine produced in the SHMT-catalyzed reaction also participates in the consequent repression of SHMT gene transcription purine biosynthesis providing two carbons and a nitrogen to [268,269]. the ring. Elevated SHMT activity has been shown to be coupled to the increased demand for DNA synthesis in rapidly PLP-Dependent Enzymes Involved in the Trans-Sulfuration proliferating cells, particularly tumor cells, like human Pathways leukaemic cells [252] and a variety of solid tumour tissues [253]. Evidence for a preferential channelling of serine towards The sulfur-containing amino acids L-, L- nucleotide biosynthesis has been found in hepatic tumors [253- and L-methionine are metabolically linked via 255]. This is obtained by an increased activity of the enzymes the trans-sulfuration and reverse trans-sulfuration pathways involved in serine biosynthesis, paralleled by the retention of (Scheme 9). In trans-sulfuration, employing the sequential SHMT and the simultaneous deletion of and action of cystathionine g-synthase and cystathionine b-lyase, L- serine aminotransferase, responsible for serine degradation. It is cysteine condenses with activated L- to form the therefore not surprising that SHMT has been often indicated as intermediate L-cystathionine, which, subsequently, is split an interesting target for cancer chemotherapy [256,257]. symmetrically into L-homocysteine and pyruvate. However, among the three members of the thymidylate cycle, Homocysteine can be methylated to yield L-methionine. SHMT is the only enzyme not yet thoroughly studied as a target Different organisms possess distinct trans-sulfuration enzymes: for cancer chemotherapy. Drugs targeting the other two plants and microbes employ only the forward pathway from enzymes, thymidylate synthase and dihydrofolate reductase, cysteine to methionine, mammals exhibit only the reverse trans- have been successfully used clinically [258,259]. Analogues of sulfuration pathway, using cystathionine b-synthase and dihydrofolate, such as methotrexate, are potent inhibitors of cystathionine g-lyase, while fungi support trans-sulfuration in both directions. In addition, some anaerobic microorganisms

NH2 H COOH Cl NH2 Cl O NH3+ N -OOC N S F H2N N OH O N OH Cl O 3-Chloro-4-(4-(2-chloro-4-(4,6-diamino- -2,2-dimethyl-s-triazin-1(2H)-yl)phenyl)- 4-Chloro-L-threonine Mimosine -butyl)benzenesulfonyl fluoride Fig. (13). Chemical structures of serine hydroxymethyltransferase inhibitors. 1310 Current Medicinal Chemistry, 2007, Vol. 14, No. 12 Amadasi et al.

O-Activated homoserine Pyruvate + Ammonia RO COO- COO- C

+ NH3 NH3 + Bacteria and plants O a-Ketobutyrate CBL S-Adenosyl methionine CGS Ammonia cystalysin or Me-cobalamin Methanethiol

MGL

Cysteine Cystathionine Homocysteine Methionine + COO- NH3 - - Fungi HS COO- HS COO S COO - + OOC S NH3 + NH + + NH3 3 NH3

CGL CBS COO- - COO HO Mammals C NH + + 3 NH3 O

Serine a -Ketobutyrate + Ammonia Scheme 9. Trans-sulfuration and reverse trans-sulfuration enzymes. are able to further process methionine using methionine g- ester for plant enzyme [271]) and L-cysteine. CGSs from lyase. bacterial sources have been studied in more detail than those from plants because the bacterial recombinant enzyme is more easily expressed in The physiological - Cystathionine -Synthase Escherichia coli. g replacement reaction follows a complex mechanism that is ping- Cystathionine g-synthase (CGS; E.C. 2.5.1.48) catalyzes the pong when [L-cys]KL-cys first reaction in methionine biosynthesis. Methionine is an [272,273]. CGS can catalyze as side reactions a g-elimination essential amino acid that can only be synthesized in reaction [274,275] as well as b-elimination, b-replacement and microorganisms and plants. Enzymes of the methionine a - or g-proton exchange reactions [274,275]. biosynthesis pathway are therefore attractive targets for the CGS is homotetrameric (dimers of dimers) with active sites design of new antibiotics and herbicides. CGS synthesizes that are situated at the subunit interface and are formed by cystathionine through a g-replacement reaction involving an residues from both subunits [276]. The structures of CGS in its activated form of homoserine (O-acetyl or O-succinyl ligand-free, internal aldimine form have been solved from E. homoserine for microbial enzyme [270], homoserine phosphate coli [277] and from Nicotiana tabacum [278]. An approach for

O COOH COO H COO- +H N 2- 3 HC O3P NH + + 3 NH3+ NH3 L-Aminoethoxyvinylglycine Propargylglycine DL-E-2-Amino-5-phosphono- -3-pentenoic acid

O S COOH N 2-O P 3 N

COOH

N Cl

3-(Phosphonomethyl)pyridine- 5-Carboxymethylthio-3-(3'-chlorophenyl)- -2-carboxylic acid -1,2,4-oxadiazole Fig. (14). Chemical structures of cystathionine g-synthase inhibitors. PLP-Dependent Enzymes as a Drug Target Current Medicinal Chemistry, 2007 Vol. 14, No. 12 1311 the development of specific and efficient inhibitors is to Cystathionine -Lyase exploit the catalytic mechanism of the target enzyme. In trans- sulfuration enzymes common compounds tested as inhibitors Cystathionine b-lyase (CBL; E.C. 4.4.1.8) is a key enzyme in are L-aminoethoxyvinylglycine (AVG, an antimicrobial amino sulfur metabolism and catalyzes the penultimate step in the acid first isolated from Streptomyces sp.) and propargylglycine microbial and plant methionine biosynthesis. The enzyme from (2-amino-4-pentynoic acid) (Fig. (14)). Both derivatives are various bacteria (including Escherichia coli [287], Salmonella able to bind at the formyl moiety of PLP and form a Schiff base typhimurium [288], Bordetella avium [289]) and plants that can be processed until being transformed into a ketimine (including Arabidopsis thaliana [290] and Spinacia oleracea derivative, which cannot proceed further and thus inactivates [291]) has been isolated and characterized. Cystathionine is the enzyme. However, these mechanism-based inhibitors do not cleaved by CBL to produce L-homocysteine, pyruvate and exhibit specificity for only one of the trans-sulfuration ammonia. CBL shows extensive homology with CGS and both enzymes. An irreversible mechanism-based inactivation of CGS proteins belong to the fold type I of PLP-dependent enzymes can be achieved with the unnatural amino acid propargylglycine [292,293]. The b-elimination reaction involves a [279,280]. This compound exerts a phytotoxic effect [281,282] transaldimination step between the substrate cystathionine and but acts, like AVG [283-285], also on other members of the g- the enzyme-bound PLP, followed by the a -proton abstraction to family, e.g. human liver cystathionine g-lyase (CGL) [285]. In yield an a -carbanion. Subsequent elimination of homocysteine the case of CGS this is due to the similarity of the catalytic generates the PLP derivative of aminoacrylate, which undergoes mechanism to that of CGL and to the limited knowledge about reverse transaldimination to produce the enzyme-bound PLP the specificity determining factors. and iminopropionate. The latter is then hydrolyzed to pyruvate and ammonia. CBL from various sources displays relatively In view of the development of new inhibitors and/or drugs, broad substrate specificity toward sulfur-containing CGS was complexed with various substrate analogs N. tabacum aminoacids. In particular, CBL from B. avium utilizes L-cystine [286] and the resulting structures were examined. In particular, as a substrate to produce L-thiocysteine [289]. The transfer of the compounds used were: DL-E-2-amino-5-phosphono-3- sulfane-sulfur from L-thiocysteine to metabolically important pentenoic acid, 3-(phosphonomethyl)-pyridine-2-carboxylic enzymes within or at the surface of sensitive eukaryotic cells is acid, 5-carboxymethylthio-3-(3’-chlorophenyl)-1,2,4-oxadiazol responsible for the cytotoxicity of this enzyme [289]. (Fig. (14)). DL-E-2-amino-5-phosphono-3-pentenoic acid is a phosphono analog of the substrate homoserine phosphate and The three-dimensional structure of E. coli and A. thaliana binds to the PLP moiety forming an external aldimine that CBL has been resolved by X-ray crystallography [294,295]. could not be isolated since it presumably undergoes a Both enzymes are tetramers constructed as dimers of dimers. tautomerization to a ketimine, as dead-end product [286]. Each monomer comprises three structurally and functionally distinct regions: an N-terminal domain, a large PLP-binding However, it shows a low affinity (Ki=27 mM) and, therefore, is unsuitable as a drug. Like DL-E-2-amino-5-phosphono-3- domain, containing most of the catalytically important pentenoic acid, 3-(phosphonomethyl)pyridine-2-carboxylic residues, and a smaller C-terminal domain. acid interacts with both the phosphate and carboxy-binding Since CBL is involved in microbial and plant methionine sites of CGS [286], but cannot form an external aldimine and its biosynthesis, it represents an attractive target for the Ki rises to 0.2 mM [286], making this compound not feasible as development of novel antimicrobial agents or herbicides. CBL a precursor for high affinity inhibitors. Instead, 5- is inhibited by the active site-directed suicide irreversible carboxymethylthio-3-(3’-chlorophenyl)-1,2,4-oxadiazol-CGS inhibitor b,b,b,-trifluoroalanine [296]. The mechanism complex [286] (Fig. (15)) reveals a unique binding mode that proposed involves the formation of a Schiff base between the accounts for the higher affinity (Ki = 2 mM) of this compound inhibitor and the enzyme-bound PLP. Subsequent elimination and provides guidelines for further drug development. The of HF from Cb produces an a ,b-unsaturated imine which is an inhibitor binds to three points of the active site, disrupting the activated Michael acceptor. This adduct can react with a stacking of PLP ring with an aromatic side chain of tyrosine: the nucleophile at the active site leading to covalent binding and carboxy- and phosphate recognition sites and the hydrophobic inactivation of the enzyme. The elucidation of the atomic binding pocket. Combining the 5-carboxymethylthio-3-(3’- structure of CBL bound to b,b,b,-trifluoroalanine confirmed the chlorophenyl)-1,2,4-oxadiazol interaction mode with binding inactivation mechanism and proved that the PLP-binding lysine to the phosphate-recognition pocket seems promising for the is the base reacting with the inhibitor via Michael addition development of high affinity inhibitors specific for plant CGS. [294,296]. CBL is also inhibited by AVG (Fig. (14)) [284]. The interaction of CBL from E. coli and A. thaliana with AVG has been studied by spectroscopic, kinetic and crystallographic methods [284,294,296]. Inhibition of CBL by AVG is reversible, but obeys a slow-binding two-step mechanism. The inhibitor binds to the free enzyme to form a weak enzyme- inhibitor complex which slowly converts to a tightened enzyme-inhibitor complex (Ki=1.1 mM, [284]). By analogy with the substrate cystathionine, AVG enters the active site and forms the external aldimine with PLP. Then, the PLP-binding lysine abstracts the a -proton from the inhibitor molecule and transfers it directly to the C4’ of the cofactor. Due to the inability of CBL to catalyze the transamination of AVG, the resulting PLP-ketimine is stable and slowly dissociates. It should be noted that L-methoxyvinylglycine (Fig. (16)), an analog of AVG missing the terminal aminomethyl part, does not inactivate CBL but undergoes a deamination reaction [284]. Fig. (15). Structure of the active site of cystathionine g-synthase from This result highlights the importance of the terminal amino Nicotiana tabacum complexed with 5-carboxymethylthio-3-(3’- group of AVG for the mechanism of inhibition. chlorophenyl)-1,2,4-oxadiazol (CTCPO) (PDB code 1I48) [286]. 1312 Current Medicinal Chemistry, 2007, Vol. 14, No. 12 Amadasi et al.

HO inhibitors that can discriminate between the homologous active O COOH sites of CGL, CGS and CBL. Any prospective inhibitor should O COOH be tested for its reactivity toward CGL, in order to exclude + +H3N adverse effects in humans [302]. On the other hand, recent data NH3 suggest that, in the rat, part of the hypotension associated with + L-Methoxyvinylglycine Rhizobitoxine NH3 haemorrhagic shock is due to the overproduction of endogenous H2S by CGL and CBS. Therefore, it has been Fig. (16). Chemical structures of cystathionine b-lyase inhibitors. proposed that CGL inhibitors, reducing H2S concentration in Rhizobitoxine (Fig. (16)) is a close analog of AVG with an the blood, could provide a novel approach to the treatment of additional CH2OH group at the distal part of the molecule that the haemorrhagic shock [303]. behaves as an antibacterial and phytotoxic amino acid [297- AVG (Fig. (14)) is a slow-tight binding inhibitor for CGL 300]. Although experimental evidence is limited, the CBL- and the mechanism of inhibition is analogous to that proposed rhizobitoxine interaction resembles that of AVG with CBL and, for CBL. It should be noted that the inhibition constant of the thus, is likely to follow the same reaction mechanism. Kinetic AVG-CBL complex is 10-fold lower than that of the AVG-CGL parameters and X-ray data of the rhizobitoxine-CBL complex complex (10.5 mM [285]). However, the inhibition constant for have not been determined due to difficulties in the preparation the AVG-CGL complex is about two orders of magnitude smaller and/or isolation of the toxin [284]. However, a model of the than the Michaelis constant for the natural substrate L- rhizobitoxine-CBL adduct was built and strongly suggests an -8 cystathionine [285]. Therefore, AVG seems to be unsuited as a interaction similar to that of AVG [284] with a Ki=2.2•10 M, lead compound for the development of antibiotics or confirming preliminary kinetic studies [297]. herbicides. Very recently, a high-throughput screening of CBL against Propargylglycine (Fig. (14)) and b,b,b,-trifluoroalanine are 50000 small molecules has identified two classes of inhibitors, suicide inhibitors of CGL [285]. The postulated mechanism of mixed steady-state inhibitors and covalently-bound inhibitors. action of propargylglycine involves an initial a -proton The three-dimensional structure of one compound belonging to abstraction by the PLP-binding lysine and the subsequent the latter class has allowed to design a small library of formation of a ketimine intermediate by reprotonation at C4’ analogues that exhibit a higher degree of inhibition [439]. (Scheme 10). Then, the lysine residue abstracts the proton at the b-position producing an allene which is in conjugation with the Cystathionine -Lyase ketimine. The latter is then capable of Michael addition to form a covalent CGL-propargylglycine complex through its activated Cystathionine g-lyase (CGL; E.C. 4.4.1.1.) catalyzes the g-carbon atom. On the basis of this mechanism, it is not second step in the reverse trans-sulfuration pathway, i.e. the surprising that inactivation of CGL by propargylglycine is a cleavage of L-cystathionine to L-cysteine, a -ketobutyrate and very fast process, since it involves an attack to the g-carbon. The ammonia. While CGL from yeast and rat liver shows an mechanism proposed for the inactivation of CGL by b,b,b,- appreciable CBL-like activity, human CGL displays a strong trifluoroalanine is similar to that proposed for the inactivation substrate specificity and a clear preference of C-S over S-S bond of CBL by the same molecule and the inhibition constant was breakage [285]. In humans, this enzyme is involved in various found to be 0.27 mM [287]. This inhibitor has a reactive b- metabolic disorders (cystathioninuria, cystinosis and carbon, which in CBL is protonated by the PLP-binding lysine. homocystinuria [301]), potentially resulting in mental or However, in CGL this residue is in an unfavorable orientation to physical impairment. react at the b-carbon. This could explain the slower inactivation -1 As revealed by the crystal structure of CGL from yeast [276], reaction of CGL by b,b,b,-trifluoroalanine (kinact=0.027 min ) the protein is a tetramer and the overall architecture is very with respect to propargylglycine [285]. similar to other related PLP-enzymes of Cys-Met metabolism, like CBL [294,295]. However, in CGL the substrate docks at the Methionine -Lyase active site in a different orientation with respect to CBL. In fact, the sulfur atom is in the gamma-position in the case of CBL and Methionine g-lyase (E.C. 4.4.1.11) catalyzes the breakdown in the delta-position in the case of CGL. of methionine by an a ,g-elimination reaction to yield a - Because different organisms display different trans- ketobutyrate, ammonia, and methanethiol [304,305]. The sulfuration enzymes, the enzymatic components in plants and enzyme has been found in many microorganisms, such as microorganisms are attractive targets for the development of Pseudomonas spp. and Aeromonas spp. and also in anaerobic antibiotics and herbicides. However, it is important to design strains. Many of these are important pathogens, including bacteria causing botulism (Clostridium botulinum), colitis

+H N Lys-E H2N Lys-E 3 H COO- COO- COO- BH+ NH+ NH+ B NH+ B: -HO P -HO3P O- -HO3P O- 3 O- O O O

N+ CH3 N+ CH3 N+ CH3 H H H External aldimine Allene Covalent adduct Scheme 10. Proposed mechanism of cystathionine g-lyase inactivation by propargylglycine. PLP-Dependent Enzymes as a Drug Target Current Medicinal Chemistry, 2007 Vol. 14, No. 12 1313

(Clostridium difficile), tooth decay (Porphyromonas Spp.), and proposed for CBL. Upon substrate binding, the Michaelis postoperative intra-abdominal infections (Bacteroides spp.) complex is rapidly converted into the external aldimine, [306]. There are also widespread anaerobic parasites of humans. followed by the abstraction of the Ca proton of the substrate to These include Entamoeba histolytica, which causes amoebiasis, yield a quinonoid intermediate. Subsequent elimination of H2S and Trichomonas vaginalis, which causes the sexually generates the PLP derivative of aminoacrylate. Protonation of transmitted disease trichomoniasis. The latter is highly the aminoacrylate and reverse transaldimination form prevalent in women and appears to be a risk factor for human iminopropionate and regenerate the enzyme-bound PLP. immunodeficiency virus infections. Methionine g-lyase has no Hydrolysis of iminopropionate to pyruvate and ammonia counterpart in mammals and so appears to be a good drug target. probably occurs outside the active site [27,316]. Recently, The 3D structure of the enzyme from T. vaginalis has been residues involved in the catalysis and intermediates of the solved both in the ligand-free form and in the complex with reaction have been identified [317,318]. Moreover, stopped- propargylglycine (Fig. (14)). The enzyme from Pseudomonas flow and quench-flow analysis of the a ,b-elimination catalyzed putida exists as homotetramer [307]. The spatial fold of by cystalysin have allowed to establish that product release is subunits, with three functionally distinct domains, and their the rate-limiting step of the catalytic pathway [318]. quarternary arrangement, is similar to those of CBL and CGS Additionally, it has been demonstrated that cystalysin has a from Escherichia coli. No structures of complexes of the high catalytic versatility and is able to catalyze the Pseudomonas enzyme with inhibitors have been determined. racemization and transamination of both enantiomers of alanine Recently, important hints were obtained on a possible pro- [319,320], the b-desulfination of L- and drug that is activated by the parasite-specific enzyme and has the b-decarboxylation of L-aspartate and oxalacetate [321]. anti-trichomonal activity in vivo. The fluorine-substitution- The virulence potential of T. denticola is strongly depen- containing analogue of methionine, trifluoromethionine, may dent on the catalytic activity of cystalysin. Its inhibition can be a substrate for methionine g-lyase, catalyzing an a,g- cause the loss of haemolytic activity and the subsequent elimination reaction to yield a -ketobutyrate, ammonia, and shortage of nutrients, resulting in limited growth and cell death. trifluoromethanethiol. This last compound is unstable under Thus, cystalysin could be an interesting target for the rational physiological conditions and nonenzymatically breaks down to design of specific inhibitors that can function as novel carbonothionic difluoride, a potent cross-linker of primary antibiotics against periodontitis. At present, only AVG (Fig. amine groups [308,309]. This compound is highly toxic to the (14)) has been found to inactivate cystalysin and the atomic pathogen in which it is produced, whereas toxicity to a structure of the protein incubated with AVG shows the presence mammalian host is minimal, as activation of the pro-drug does of a ketimine PLP-AVG covalent adduct, held in place mainly by not occur in the absence of methionine g-lyase. Thus, pathogen- hydrogen bonds and hydrophobic interactions [27]. This generated carbonothionic difluoride is bound by the pathogen cofactor-inhibitor complex is similar to that observed with CBL material before it encounters any host molecules. after interaction with AVG and an analogous inactivation Trifluoromethionine is efficacious in vitro and in vivo against mechanism has been proposed [284]. However, the analysis of microorganisms containing methionine g-lyase, representing a the ligand binding pocket reveals that cystalysin displays a lead compound for a novel class of drugs against a range of higher accessibility to the PLP cofactor, which indicates the anaerobic pathogens [310,311]. possibility to accommodate bulky substrates and inhibitors.

Cystalysin Cystathionine -Synthase Cystalysin is a PLP-dependent enzyme isolated from Cystathionine b-synthase (CBS; EC 4.2.1.22) catalyzes the Treponema denticola which catalyzes the a ,b-elimination of L- condensation of serine and homocysteine to form cystathionine cysteine to produce pyruvate, ammonia and H S [312,313]. 2 via a b-replacement reaction. CBS from higher eukaryotes is a T. denticola, a major member of the oral spirochetes, is a unique PLP-dependent enzyme, which contains a domain for putative pathogenic agent in adult periodontitis, acute binding a heme and a domain for binding AdoMet. The role of necrotizing ulcerative gingivitis and juvenile and human the heme as well as that of AdoMet is to regulate the enzyme immunodeficiency virus-related periodontal diseases. activity, without directly participating in the catalytic action Cystalysin is endowed with hemolytic and hemoxidative [322,323]. The human full-length CBS structure has not been activities, both probably dependent on the production of H2S, determined. However, two crystallographic structures of the which is toxic for most cells. As a consequence of lysis, various truncated enzyme, containing the heme domain, have been nutrients contained in the cytoplasm of erythrocytes become reported [323,324]. Recently, the AdoMet binding site has been available to the bacterium, in particular the iron of the heme found to be shaped by two hydrophobic domains, named CBS1 group. In addition, the elevated H2S concentrations created in and CBS2, located within the C-terminal region of CBS the periodontal pocket generate an ecological niche for T. [325,326]. CBS is structurally related to the fold type II of the denticola. Thus, cystalysin can be classified as a novel PLP- PLP-dependent enzymes. dependent virulence factor [313-315]. CBS represents a key enzyme in one of the two major The atomic structure of cystalysin, solved at 1.9Å¢ pathways for the clearance of intracellular homocysteine. More resolution, reveals that the protein is a homodimer belonging to then 100 mutations have been described in CBS gene, and the fold type I group [27]. Each monomer folds into two metabolic dysfunctions due to CBS mutations represent the domains: the large domain which carries the PLP cofactor, and most common cause of hereditary homocystinuria, a disease the small domain, made up of the two terminal regions of the which is characterized by skeletal problems, dislocated eye polypeptide chain [27]. Biochemical studies on the PLP lenses, vascular problems and mental retardation [327]. binding mode of cystalysin have shown that the coenzyme is Moreover, it is well established that homocysteine is an bound at the active site in two different forms and that the independent risk factor, as smoking or hyperlipidemia, for enzyme possesses a broad substrate specificity being a atherosclerotic, cardiovascular, cerebrovascular and peripheral cyst(e)ine C-S lyase [316] rather than a cysteine desulfydrase. vascular diseases, and for deep vein thrombosis and On the basis of the active site architecture, a catalytic thromboembolism [328]. It follows that there is a high interest mechanism has been proposed for the a ,b-elimination of L- in gaining insights into CBS catalytic mechanism and its cysteine catalyzed by cystalysin similar to the mechanism regulation. This should allow a better understanding of its 1314 Current Medicinal Chemistry, 2007, Vol. 14, No. 12 Amadasi et al. physiological role and, if possible, the design of allosteric Several inhibitors of PLP-dependent enzymes were tested for effectors, able to enhance the activity of the enzyme, that might inhibitory activity against CS from the leaves of Echinochloa eventually be applied in the therapy of cardiovascular diseases. crus-galli [345,346]. Preincubation with DL-allylglycine, DL- On the other hand, very recently, the use of CBS inhibitors has propargylglycine, b-chloro-L-alanine, 3-bromopropionate, been proposed as a possible novel therapeutic approach to the amino-oxyacetate, hydroxylamine and cycloserine, at treatment of acute stroke. In fact H2S, an important mediator of concentrations in the millimolar range, inhibited the CS ischemic damage, seems to be produced from cysteine in the activity. This finding was correlated with in vivo phytotoxicity, cerebral cortex through the action of CBS [329]. suggesting that inhibition of CS might affect the growth of E. crus-galli. However, the supplementation of hexogenous cysteine failed to reverse inhibition in test tube bioassays, O-Acetylserine Sulfhydrylase suggesting that growth inhibition could not be entirely O-acetylserine sulfhydrylase (OASS, O-acetylserine attributed to the starvation of cysteine [345]. Inhibition studies (thiol)lyase; EC 2.5.1.47), belongs to the fold type II of the on plant OASS by b-substituted alanyl inhibitors were also PLP–dependent enzymes. OASS catalyzes the last reaction in the carried out [347]. biosynthesis of cysteine in bacteria, archaea and plants, a b- replacement reaction that leads to the formation of cysteine from O-acetylserine (OAS) and sulfide, through a Bi Bi ping- pong kinetic mechanism [330]. Two isoenzymes, OASS-A and OASS-B, are present in several bacteria and exhibit different substrate specificities [331]. Although humans lack OASS, cysteine is not an essential aminoacid, since it can be synthesized upon condensation of serine and homocysteine, catalyzed by CBS, to yield cystathionine, that is deaminated and cleaved by CGL forming cysteine and a -ketobutyrate. In vivo, OASS forms bi-enzyme complexes with ATP sulfurylase and serine acetyltransferase (SAT), the preceding enzyme in the cysteine biosynthetic pathway. The complex with SAT is referred to as cysteine synthase (CS). Upon formation of CS, OASS activity is reduced both in bacteria [332,333], and plants [334]. The stability of the complex is related to sulfur availability in the cell. OAS accumulation favors complex dissociation, an event that is prevented by the presence of Fig. (17). Structure of the active site of 0-acetylserine sulfhydrylase-A sulfide [332,334-336]. from Haemophilus influenzae complexed with the C-terminal decapeptide The structure of the OASS homodimer has been solved for of serine acetyltransferase (PDB code 1Y7L) [340]. the A isozyme from Salmonella typhimurium [337-339], Inhibition of CS as a therapeutic strategy is of particular [340] and from the plant Haemophilus influenzae Arabidopsis interest in the case of Trichomonas vaginalis, an anaerobic , and for the B isozyme from [341]. In thaliana Escherichia coli protozoan parasite of humans [311]. T. vaginalis lacks particular, the determination of the structure of S. typhimurium and relies on cysteine as a major redox buffer. It OASS in the absence [337] and presence of substrate analogues also seems to lack SAT, the enzyme producing OAS, and all four and allosteric effectors [339] indicated that the formation of the enzymes of the forward and reverse trans-sulfuration pathway, external aldimine of PLP with L-cysteine, L-serine and suggesting that direct methionine-cysteine interconversions do methionine is associated with a conformational change from an not occur [311]. It thus appears that cysteine biosynthesis open (inactive) to a closed (active) structure. Binding of involves CS utilizing O-phosphoserine, rather than OAS, and chloride to an allosteric anion-binding site, located at the dimer sulfide or thiosulfate as substrates. interface, stabilizes a conformation that differs both from the open conformation of the internal aldimine and the closed one The somewhat broad substrate specificity of OASS allows of external aldimine [338]. In such state the formation of the technological applications in the production of novel b– external Schiff base and, thus, the subsequent chemistry are substituted L-aminoacids to be used as building blocks for the inhibited. The authors suggested that the chloride ion, or other synthesis of pharmaceuticals and agrochemicals [341,348,349], anions, such as sulfate [342], may behave as an analog of like the S-phenyl-L-cysteine contained in the HIV-protease sulfide, the physiological inhibitor of OASS. inhibitor Viracept [350]. The availability of structural data for wild type OASS and some mutants, from different sources, can be exploited for the Cysteine S-Conjugate -Lyases structure-based design of specific inhibitors, since Some PLP-dependent enzymes, mainly aminotransferases, microspectrophotometric studies demonstrated the catalytic are able to catalyze cysteine S-conjugate b-lyase reactions. The competence of OASS in the crystalline state [343]. Inhibition of products are pyruvate, ammonia and a sulfur-containing OASS, an enzyme present in bacteria and plants, but not in fragment [351]. Different effects of these kind of side reactions vertebrates, features possible applications in the design of have been reported. If the sulfur-containing fragment is reactive, herbicides and antibiotics. In principle, this goal could be the reaction may determine bioactivation of toxicants, as tackled by at least three different approaches: i) specific reported for halogenated alkenes and cysplatin [351,352]. On inhibition by substrate or transition state analogues, ii) use of the other hand, targeting of prodrugs has been proposed as a allosteric regulatory molecules, iii) interference with possible beneficial effect of the cysteine S-conjugate b-lyase interprotein interactions upon CS complex formation. The latter action. For instance a bioactivation mechanism based on the approach has been recently undertaken, triggered by the cysteine S-conjugate b-lyases has been proposed to explain the observation that the C-terminal decapeptide of SAT behaves as a anti-cancer properties of allium-derived compounds [353] and competitive inhibitor of OASS, binding to the a -carboxyl has been hypothesized as a route to target pharmacologically subsite (Fig. (17)) [340] with dissociation constants in the active selenol compounds to the kidney [354]. hundreds of nanomolar range [336,344]. PLP-Dependent Enzymes as a Drug Target Current Medicinal Chemistry, 2007 Vol. 14, No. 12 1315

PLP-Dependent Enzymes Involved in Heme and Essential formation of 4,5-diaminovalerate as intermediate, which can Amino Acid Synthesis dissociate from the enzyme, leaving the latter in the free PLP form and slowing down the reaction. It is not clear whether this Glutamate-1-Semialdehyde-1,2-Aminomutase detrimental dissociation, which is clearly detectable during the Glutamate-1-semialdehyde-1,2-mutase (or glutamate 1- catalyzed reaction in vitro [365], is an essential feature of the semialdehyde aminomutase; GSAM; E.C. 5.4.3.8) catalyzes the catalytic mechanism and if it actually takes place in vivo. isomerisation of glutamate-1-semialdehyde (GSA) into 5- Some inhibitors of PLP-dependent enzymes, such as aminolevulinic acid, the universal precursor of tetrapyrroles, gabaculine (Fig. (18)), g-ethynyl GABA and g-vinyl GABA have such as chlorophyll, heme and coenzyme B12 . This enzyme is been experimented on GSAM [366,367]. However, these only present in plants, algae and most bacteria, which obtain compounds do not possess the specificity and efficiency GSA from the reduction of glutamyl-tRNA, catalyzed by required to act as antibacterial agents. Recently, the reaction of a glutamyl-tRNA reductase [355]. In animals, fungi and some novel and efficient inhibitor, specifically designed for GSAM, bacteria, 5-aminolevulinic acid is synthesized directly through 2,3-diaminopropyl sulfate (Fig. (18)), was analysed [368]. The the condensation of glycine and succinyl-CoA, a reaction inhibition, which was characterized for the enzyme from catalyzed by another PLP-dependent enzyme, 5-aminolevulinic Synechococcus, takes place upon the elimination of the sulfate acid synthase [356]. For this reason, GSAM is an interesting group, followed by the nucleophilic attack of an unidentified target for specific antibacterial agents. The essential role of the residue of the enzyme. enzyme in metabolism has been demonstrated for important pathogens such as [357], Staphylococcus aureus Salmonella typhimurium [358] and Pseudomonas aeruginosa [359]. The three-dimensional structure of GSAM, which has been Threonine, as well as tryptophan, is among the essential solved from three different sources [360-362], and the catalytic aminoacids; thus, vertebrates depend for its supply on food mechanism strictly resemble those of the aminotransferases uptake. Threonine synthase (THS; E.C. 4.2.99.2) and tryptophan [76]. Interestingly, Moser et al. [363], upon the resolution of synthase (see below) are PLP-dependent enzymes only present glutamyl-tRNA reductase crystal structure, proposed a model in plants, bacteria and fungi, making them potential candidates whereby the large void of the V-shaped enzyme may be for compounds acting as antibiotics or pesticides and occupied by GSAM. According to this hypothesis GSA, which is herbicides [369,370]. THS catalyzes a b,g- replacement reaction an unstable compound at neutral pH, might be produced at the of O-phospho-homoserine yielding threonine and inorganic active site of the reductase and directly channelled to GSAM, phosphate. Competitive inhibition was observed for allowing for the efficient synthesis of 5-aminolevulinic acid. A homophosphoserine analogues with Ki in the micromolar range recent crystallographic study on GSAM suggested the presence [371,372], whereas irreversible inhibition was observed for DL- of a gating loop which controls accessibility to the active site 2-amino-3-[(phosphonomethyl)amino)]propanoic acid and Z-2- and a cross-talk mechanism between the two subunits that amino-5-phosphono-3-pentenoic acid, with rate of inactivation might be involved in substrate channelling and catalysis [364]. of the order of 1.5 min-1 and a 1:1 stoichiometry [372]. The proposed mechanism of inactivation differs for the two O COOH inhibitors. For the case of DL-2-amino-3-[(phosphonomethyl) H O S O- amino)]propanoic acid, upon the sequential formation of the external aldimine and the quinonoid intermediate, the electron H N O 2 NH2 pair of the 3-amine substituent may attack the pyridine ring to H 2N give a stable spirocyclic derivative of PLP [372] (Scheme 11). In the case of Z-2-amino-5-phosphono-3-pentenoic acid, the 2,3-Diaminopropyl sulfate Gabaculine reaction proceeds with formation of an equilibrium between two quinonoid species, one of which is particularly stable. However, Fig. (18). Chemical structures of glutamate 1-semialdehyde aminomutase the lack of radiolabeled inhibitors makes it difficult to rule out inhibitors. an alternative mechanism involving a covalent enzyme The distinctive feature of GSAM with respect to other modification [372]. The 3D structure of the enzyme has been aminotransferases is that it catalyzes an intramolecular determined from Thermus thermophilus [373], Saccharomyces exchange of the carbonyl and amino groups, which are both cerevisiae [374] and Arapidopsis thaliana [375,376]. present on the substrate GSA. The catalytic cycle begins with Interestingly, the activity of THS from A. thaliana is GSA and the cofactor as pyridoxamine 5'-phosphate and is allosterically modulated by S-adenosylmethionine via a completed without the involvement of a second amino acid conformational change that leads to a reorganization of the substrate. A key event in the reaction mechanism is the active site and a reorientation of PLP [376]. Furthermore,

:Base-E

O H O O- COO- 2- 2- O P N O3P N O- 3 H H NH+ 2- NH+ NH+ O3P N 2- 2-O P 2-O P O3P O- 3 O- 3 O- O O O

N CH N+ CH3 3 N CH3 H H H External aldimine Quinonoid intermediate Spirocyclic derivative

Scheme 11. Proposed mechanism of threonine synthase inactivation by DL-2-amino-3-[(phosphonomethyl)amino)]propanoic acid. 1316 Current Medicinal Chemistry, 2007, Vol. 14, No. 12 Amadasi et al. structures of THS from Mycobacterium tuberculosis and from Mycobacterium tuberculosis complexed with the same Escherichia coli have been determined and coordinates inhibitors were built as the first step in developing specific deposited in the Protein Data Bank, without any accompanying drugs against tuberculosis [399]. publication. These THS structures open the way to studies Studies on the regulation of TrpR, the regulator of aimed to develop new reversible as well as irreversible tryptophan metabolism, and trpA and trpB, the genes for TS inhibitors, eventually candidates for antibiotic and herbicide subunits in Chlamydia trachomatis, have unveiled distinct activity. functional properties for TS present in C. trachomatis serovars occurring in ocular and genital infections [400,401]. Tryptophan Synthase Furthermore, TS has been found to be one of the enzymes involved in aminoacid biosynthesis that shows an increased Bacterial tryptophan synthase (TS; E.C. 4.2.1.20) is an a 2b2 expression in Phytophthora infestans during biotrophic and complex that catalyzes the last two steps in the biosynthesis of necrotrophic infection phases [402]. Interestingly, compounds L-tryptophan [25,377,378]. In yeast and fungi the a - and b- that act in plant defence mechanisms are synthesized in a series subunit constitute a single polypeptide chain. Indole-3- of reactions, including the production of indole by a specific glycerol phosphate is cleaved in the a -active site to generate a subunit of TS that is not devoted to produce tryptophan. glyceradehyde-3-phosphate and indole. The latter is Overall, these findings indicate that both a and b subunits are subsequently channelled via a hydrophobic tunnel to the b- suitable targets for the development of compounds active as active site, that contains PLP bound via a Schiff base, where it is pesticides, herbicides or antibiotics. combined with L-serine to form L-tryptophan. This channelling avoids the escape of indole to solution and represents the first detected example of intramolecular substrate transfer [379]. The reaction proceeds through the formation of several intermediates, characterized by distinct absorption and emission properties. The a - and b-subunit activities are reciprocally regulated through the stabilization of a catalytically inactive "open" state or a catalytically active "closed" state of both the a and b subunits [380-382]. Depending on the catalytic intermediate present in the active site, regulatory signals are generated that affect the opposite subunit, thus finely tuning the subunit catalytic activities and keeping them in phase. The structure of the wild type TS and several mutants has been determined [379,383-392] and function has been assessed in the crystalline state by polarized absorption microspectrophotometry in order to determine the experimental conditions for the selective accumulation of distinct catalytic intermediates, eventually suitable for X-ray crystallographic analysis [384,393-395]. These studies provide to drug designers the reassuring information on the validity of Fig. (20). Structure of the active site of tryptophan synthase from the enzyme structure for developing inhibitors. Computational Salmonella typhimurium complexed with N-[1H-indol-3-yl-acetyl] procedures have been used for the design of new inhibitors of aspartic acid (IAD) (PDB code 1K3U) [391]. the a -subunit [396]. The starting structure was that of the complex with the a -subunit ligand and allosteric effector Serine Racemase indole-3-propanol phosphate. By several structural modifications, guided by the evaluation of the interaction The PLP-dependent enzyme serine racemase converts L- energy and volume occupancy, a series of indole-acetyl serine to D-serine. The enzyme has also been shown to catalyze aminoacids were proposed as potential ligands (Fig. (19)). The b-elimination reactions, as the conversion of L-serine and L- three-dimensional structure of the a 2b2 complex in the absence serine-O-sulfate to pyruvate [403]. Significant amounts of D- and presence of some of these indole-acetyl aminoacids serine have been found in the brain. This D-amino acid is provided convincing evidence of the different roles played by synthesized in astrocytes and acts as an endogenous ligand for these a -subunit ligands [391] (Fig. (20)). Furthermore, a series the “glycine site” of N-methyl-D-aspartate (NMDA)-type of arylthioalkylphosphonate inhibitors (Fig. (19)), that are glutamate receptors, that play a central role in excitatory transition-state analogues and isosteres of the a -subunit ligand neurotransmission, neuronal plasticity, memory and learning indole propanol phosphate, were synthesised and assayed in [404]. Over-activation of NMDA receptors may lead to cell death vitro and in vivo, exhibiting micromolar IC50 s [397]. The and contributes to post-ischemic brain damage [404]. Indeed, phosphonate moiety was selected due to its capability of blockers of the D-serine binding site of the NMDA receptor were forming hydrogen bonds and not to be degraded by found to be neuroprotective in animal models of stroke [405]. phosphatases. Some of these inhibitors were complexed with TS Diminishing NMDA neurotransmission might be beneficial in and their structures determined [398]. Molecular models of TS other conditions associated with excess excitation, like neurodegenerative diseases [404,406]. Studies proposing that O D-serine and NMDA transmission may be relevant in psycotic COOH states as schizophrenia have also been reported [407]. Because OH the inhibition of D-serine production is a possible strategy to S N diminish NMDA neurotransmission, in recent years, there has H X P OH been an increasing interest on serine racemase as a potential O drug target. Strisovsky et al. analyzed a series of compounds N OH H A representative example of derived from L-serine and L-serine-O-sulfate, identifying Indole-3-acetyl aminoacids arylthioalkylphosphonate inhibitor several weak competitive inhibitors of the mouse enzyme. The authors stated that the most important structural feature for Fig. (19). Chemical structures of typtophan synthase a- subunit inhibitors. binding to mouse serine racemase is the presence of two PLP-Dependent Enzymes as a Drug Target Current Medicinal Chemistry, 2007 Vol. 14, No. 12 1317 adjacent carboxylates with optimal C-C distance of 2.6-3.2 Å PLP-Dependent Enzymes Involved in Biotin Biosynthesis [403]. Very recently, Dixon et al. applied one-bead one- compound combinatorial chemistry, together with a high- 7,8-Diaminopelargonic Acid Synthase throughput screening based on fluorescently labeled enzyme, to The biosynthesis of the vitamin biotin, a cofactor in find inhibitors of human serine racemase. Several peptides biological carboxylation reactions, occurs in microorganisms showing moderate inhibitory potency (high micromolar Kis) and plants and involves at least four different enzymes (Scheme were identified. Interestingly, these inhibitors exhibited no 12) [412,413]. Because biotin synthesis is unique to plants and structural similarities to L-serine, the natural enzyme substrate microorganisms, enzymes of this pathway are potential targets [408]. for the development of antimicrobial drugs and herbicides. One of these enzymes is 7,8-diaminopelargonic acid synthase (DAPAS; EC 2.6.1.62), an aminotransferase that catalyzes the Alliinase antepenultimate step in this pathway: the conversion of 7-keto- Garlic is well known to exhibit therapeutic effects [409]. 8-aminopelargonic acid to7,8-diaminopelargonic acid [414]. Antibacterial, antithrombotic and anticancer activities are DAPAS from Escherichia coli is a homodimer with a exhibited by sulfur-containing compounds, such as allicin, molecular mass of 94 kDa [415] and contains 429 residues per methylallyl-trisulfide and diallyl-trisulfide. In particular, monomer [416]. It is unique among aminotransferases in that it allicin is formed by the alliinase-assisted hydrolysis of S-allyl- uses S-adenosyl-L-methionine as amino group donor [415]. L-cysteine sulfoxide. Alliinase is a PLP-dependent enzyme that DAPAS is expected to follow the established overall mechanism has been exploited for the production of allicin either in vitro, of B6-dependent aminotransferases. Upon binding of S- conjugated to a Sepharose column [410], or in situ conjugated adenosyl-L-methionine, the substrate for the first part of the to a targeting monoclonal antibody [411]. This represents an catalytic cycle, an external aldimine is formed between the interesting example of the use of a PLP-dependent enzyme for substrate and the cofactor through a transaldimination process. the production of a therapeutically active molecule. The reaction proceeds through deprotonation of the former Ca O carbon of the substrate, leading to a quinonoid intermediate. This step is followed by protonation of the C4’ carbon. C COOH COOH CoAS Hydrolysis of the resulting ketimine intermediate gives the H2N ketoacid of the first substrate S-adenosyl-L-methionine and Pimeloyl CoA L-Alanine pyridoxamine phosphate bound to the enzyme by non-covalent bonds. The second half of the catalytic cycle is in principle the reversal of the first steps. 7-Keto-8-aminopelargonic acid, the second substrate, binds and receives the amino group from CoASH 7-Keto-8-aminopelargonic acid pyridoxamine phosphate and the PLP-enzyme complex is synthase regenerated. Thus, both substrates have to be accommodated in CO2 the same active site, despite differences in size and chemical properties. Although 7-keto-8-aminopelargonic acid bears an H N O 2 amino group, DAPAS uses 7-keto-8-aminopelargonic acid only C COO H as an amino group acceptor, not as an amino group donor, i.e. it is not transaminated by DAPAS [414]. Sequence and structure 7-Keto-8-aminopelargonic acid analysis suggested that DAPAS may belong to the fold type I group, which includes a number of enzymes of known structure: S-Adenosyl methionine w-aminotransferase, ornithine aminotransferase, 2,2-dialkyl- glycine decarboxylase and GSAM [417]. Several antimicrobial compounds that specifically inhibit DAPAS have been isolated 7,8-Diaminopelargonic acid from Streptomyces spp. The best studied of these compounds, synthase amiclenomycin (Fig. (21)), is particularly active against mycobacteria [418,419]. This amino acid has been isolated as a free acid and as a part of di- and tripeptides and all these S-Adenosyl-2-oxo compounds show antibiotic activities. There is evidence,

-4-methylthiobutiric acid however, that amiclenomycin acts as an enzyme irreversible inhibitor in the form of the free amino acid and that the di- and

H2N NH2 tripeptides are hydrolyzed by bacterial peptidases, which

COO H release amiclenomycin [419]. Amiclenomycin exists as cis and trans isomers (Fig. (21)). On the basis of NMR spectra it was concluded that the natural product exhibits a cis rather than a 7,8-Diaminopelargonic acid trans geometry [420]. Inhibition studies of E. coli DAPAS by amiclenomycin indicated that the inhibitor binds to the 7-keto- 8-aminopelargonic acid/7,8-diaminopelargonic acid binding site of the enzyme [421]. The crystal structures of the complexes O formed between the E. coli holoenzyme and cis and trans amiclenomycin have been determined [422]. The structure

H2N NH2 analysis revealed that the inhibitor forms a covalent adduct with the cofactor PLP, which mimics the external aldimine. The COOH proposed mechanism of inactivation suggests the formation of S a covalent adduct, which is tightly bound to the active site, and

Biotin is accompanied by aromatization of the cyclohexadiene ring of amiclenomycin (Scheme 13) [422,423]. Inhibition studies, Scheme 12. Biosynthetic pathway of biotin: reactions catalyzed by 7-keto- absorption spectra, and the crystallographic analysis have 8-aminopelargonic acid synthase and 7,8-diaminopelargonic acid provided evidence that only the cis isomer is a potent inhibitor synthase. of DAPAS [422]. 1318 Current Medicinal Chemistry, 2007, Vol. 14, No. 12 Amadasi et al.

H2N COOH H 2N COOH OH

NH2 NH2 NH2

Cis-Aminoclenomycin Trans-Aminoclenomycin 4-(4c-Aminocyclohexa-2,5-dien-1r-yl)propanol Fig. (21). Chemical structures of 7,8-diaminopelargonic acid synthase inhibitors.

It was recently reported that the gene encoding for DAPAS dien-1r-yl)propanol specifically targets DAPAS in vivo. The (bioA) is implicated in long-term survival of mycobacteria inactivation of M. tuberculosis DAPAS seems to follow the [424], making this enzyme an attractive target for the same reaction pathway of that found for the E. coli enzyme. This development of new drugs against Mycobacterium tuberculosis. mechanism is reminiscent of that proposed by Rando [425] and Amiclenomycin and a new analogue, 4-(4c-aminocyclohexa-2,5- by Silverman and coworkers [232,426,427] for the inactivation dien-1r-yl)propanol (Fig. (21)), were shown to be suicide of GABA-AT by several compounds, and more recently by substrates of M. tuberculosis DAPAS, with inactivation others for the inactivation of DAA [428], and AlaR [221] by -1 parameters of Ki = 12 mM and kinact = 0.35 min , and Ki = 20 mM DCS. It is quite interesting to note that all these PLP-dependent -1 and kinact = 0.56 min , for amiclenomycin and 4-(4c- enzymes are inhibited by a similar mechanism, ultimately aminocyclohexa-2,5-dien-1r-yl)propanol, respectively. The yielding an aromatic ring that does not dissociate from the inactivation was irreversible, and the partition ratios were 1.0 active site. Interestingly, when Mycobacterium DAPAS was and 1.1 for amiclenomycin and 4-(4c-aminocyclohexa-2,5-dien- reacted with gabaculine and the antituberculosis drug 1r-yl)propanol, respectively, which make these inactivators cycloserine, the enzyme was not inhibited even at high particularly efficient. 4-(4c-Aminocyclohexa-2,5-dien-1r- concentration (1.3 mM). yl)propanol completely inhibited the growth of an E. coli bioA mutant strain transformed with a plasmid expressing the M. 7-Keto-8-Aminopelargonic Acid Synthase tuberculosis bioA gene coding for DAPAS. Reversal of the antibiotic effect was observed on the addition of biotin or 7-Keto-8-aminopelargonic acid synthase (or 8-amino-7- diaminopelargonic acid [423]. Thus, 4-(4c-aminocyclohexa-2,5- oxopelargonate synthase, or 8-amino-7-oxononanoate

H R H R

E-Lys E-Lys H N: 2 NH + H N+ 3 N+ H H

-HO3P O- -HO3P O- O O

N+ CH3 N CH3 H H External aldimine Quinonoid intermediate

R B: BH+ H R

Enz-Lys Enz-Lys H N: 2 H H2N: N H N+ H H H H -HO P 3 O- -HO3P O- O O

N+ CH3 N+ CH3 H H Aromatic adduct Ketimine intermediate Scheme 13. Proposed mechanism of 7,8-diaminopelargonic acid synthase inactivation by amiclenomycin (adapted from [423]). PLP-Dependent Enzymes as a Drug Target Current Medicinal Chemistry, 2007 Vol. 14, No. 12 1319 synthase, KAPAS; EC 2.3.2.47) is a member of the a -oxoamine genome suggests that this enzyme may have acquired a different synthase family of enzymes, a small group of proteins that function in eukaryotes, i.e. it catalyzes a different reaction on a typically catalyze Claisen condensations between amino acids substrate possibly structurally related to threonine. Indeed, and acyl-CoA thioesters, with concomitant decarboxylation. very recently, it has been found [435] that the human enzyme KAPAS is a homodimeric PLP-dependent enzyme. Amino acid binds O-phospho-homoserine and O-phospho-threonine, sequence comparison and the 3D structure of the enzyme slowly degrading them to a -ketoglutarate and a -ketobutyrate, suggest that it belongs to the fold type I [429,430]. KAPAS respectively, phosphate and ammonia, and is unable to catalyzes the formation of 7-keto-8-aminopelargonic acid from synthesize L-threonine. These findings indicate that inhibitors L-alanine and pimeloyl-CoA, the first step of biotin of bacterial THS may also interact with the human homolog, biosynthesis (Scheme 12). Attempts to design substrate and thus adding a further level of complexity to the development of intermediate analogs as inhibitors have been reported [431]. specific, non toxic compounds with antibiotic activity. The Among these compounds, (±)8-Amino-7-oxo-8-phosphonono- potential generality of this situation for enzyme targets, nanoic acid (Fig. (22)), designed as a non-decarboxylating previously assumed to be confined in bacteria, calls for a more analog of the reaction intermediate, was found to be a reversible in-depth exploitation of the genomics information for the slow binding inhibitor of the enzyme (Ki = 7 µM). Structural selection of drug targets. Moreover, a thorough comparison of studies, that would be of great interest for the understanding of PLP-dependent enzyme genes in human genome and the inactivation mechanism, have not yet been performed. pathological species may lead to the discovery of new and Indeed, many aminophosphonate derivatives have already been specific targets. utilized as inhibitors of PLP-dependent enzymes, mainly AlaR, but none of them has been marketed clinically [226,227]. To date the structure of KAPAS from Escherichia coli has been ACKNOWLEDGMENTS determined in the apo- and holo-form [429], and as the external This work was supported by a grant from the Italian aldimine complex [432]. Furthermore, the structure of a Ministry of University (COFIN2005-2006). covalent adduct of KAPAS with the suicide inhibitor trifluoroalanine (Fig. (22)) has recently been reported [433]. It is well established that a wide range of PLP-dependent enzymes ABBREVIATIONS can be inhibited by b-halo and b-polyhalo-analogues of their substrate amino acids. However, the reported crystallographic AcOAT = N-Acetylornithine aminotransferase structure gives evidence of a difluorinated adduct, suggesting AGXT = alanine:glyoxylate aminotransferase an inactivation mechanism, involving an earlier decarboxylation step, that is quite different from the one AlaR = alanine racemase experimentally demonstrated [294] for other PLP-dependent AONS = 8-amino-7-oxopelargonate synthase enzymes with halogenated analogues [433]. AVG = L-aminoethoxyvinylglycine CF 3 H2N PO3H2 BCAAs = branched-chain amino acids -OOC NH H C COOH BCAT = branched-chain amino acid aminotransferase H 2 3 O CBL = cystathionine b-lyase CBS = cystathionine b -synthase Trifluoroalanine (±)8-Amino-7-oxo-8-phosphonononanoic acid CGL = cystathionine g-lyase Fig. (22). Chemical structures of 7-keto-8-aminopelargonic acid synthase inhibitors. CGS = cystathionine g-synthase CNS = central nervous system FROM GENOMICS TO DRUG TARGETS: THE CASE OF CS = cysteine synthase PLP-DEPENDENT ENZYME GENES DAA = D-amino acid aminotransferase The availability of the complete genome from several Dap = N-succinyl-L,L-diaminopimelate species, including Homo sapiens, has allowed the DAPAS = 7,8-diaminopelargonic acid synthase characterization of the distribution of PLP-dependent enzyme genes [31]. It was found that the PLP-dependent enzyme genes DapAT = N-succinyl-L,L-diaminopimelate amino- in archea and bacteria are about 1.5% with respect to the all transferase genes and almost two-fold less in eukaryota. Furthermore, only DapDC = diaminopimelate decarboxylase one-third of the PLP-dependent activities present in DCS = D-cycloserine Escherichia coli are also present in H. sapiens. The search method that was used identifies the conservation of structural DDC = DOPA decarboxylase motives within a defined family [434]. This approach was DFMO = a -difluoromethylornithine facilitated by the limited structural diversity of PLP-dependent enzymes, being grouped in five fold types [21]. At least a dozen DOPA = dihydroxyphenylalanine “novel” PLP-dependent enzyme genes were identified in the GABA = g-aminobutyric acid human genome. The characterization of their functional GABA-AT = g-aminobutyric acid aminotransferase properties might open the way to new drug targets or may cast doubts on the possibility of using as a drug target some PLP- GSAM = glutamate-1-semialdehyde-1,2-aminomutase dependent enzymes supposedly present only in bacteria. A HDC = histidine decarboxylase representative example is a PLP-dependent human gene highly homologous to the bacterial THS. Because threonine is an H4PteGlu = tetrahydropteroylglutamate essential aminoacid, THS should not be present in the human L-Orn = L-ornithine genome and, therefore, it was proposed as a drug target for NMDA = N-methyl-D-aspartate inhibitors with antibiotic activity. Its presence in the human 1320 Current Medicinal Chemistry, 2007, Vol. 14, No. 12 Amadasi et al.

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Received: January 01, 2007 Revised: March 12, 2007 Accepted: March 13, 2007