Inhibition of Alkaline Phosphatase

Send Orders for Reprints to [email protected] Mini-Reviews in Medicinal Chemistry, 2014, 14, 000-000

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Inhibition of Alkaline Phosphatase: An Emerging New Drug Target Mariya Al-Rashida1* and Jamshed Iqbal2,3* 1

Department of Chemistry, Forman Christian College (A Chartered University), Ferozepur Road, Lahore 54600, Pakistan; 2Centre for Advanced Drug Research; 3Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad, Pakistan Abstract: Alkaline phosphatase (AP, EC 3.1.3.1.) is a metalloenzyme that belongs to a family of ectonucleotidases. The other members of ectonucleotidase family are ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ectonucleotide pyrophosphatase/phosphodiesterases (E-NPPs) and ecto-5ʹ-nucleotidase (e5ʹNT). These ectonucleotidases are responsible for hydrolyzing extracellular nucleotides to nucleosides including adenosine. Many of these extracellular nucleotides and adenosine are important signaling molecules that act on their respective receptors (adenosine activated P1 receptor; nucleotide activated P2 receptor, each having many sub-types) and are therefore responsible for triggering cellular responses that lead to important physiological and immunological changes. A dedicated, concerted cohort of ectonucleotidases is responsible for controlling the availability of these extracellular signaling molecules at their respective receptors. Inhibitors of these ectonucleotidases provide the means by which these cellular processes can be modulated. This mini review has been written in the wake of mounting evidence of potential therapeutic benefits associated with inhibition of alkaline phosphatases and aims to provide prolific leads to design more potent and selective AP inhibitors.

Keywords: Alkaline phosphatase, tissue non-specific alkaline phosphatase (TNAP), tissue specific alkaline phosphatase, inhibitors of alkaline phosphatase, ectonucleotidase, mineralization defects. INTRODUCTION Alkaline phosphatase (AP, EC 3.1.3.1.) belongs to a family of ectonucleotidase enzymes that hydrolyze extracellular nucleotides to nucleosides including adenosine [1]. APs are substrate non-specific enzymes and a variety of phosphomonoesters are hydrolyzed by them. They are also responsible for catalyzing certain transphosphorylation reactions [2, 3]. In addition, APs are also known to hydrolyze a diverse assortment of phosphate containing compounds, such as glucose-phosphates, inorganic pyrophosphate (PPi), inorganic polyphosphates, phosphatidates, and bis(p-nitrophenyl) phosphate [1]. APs are responsible for the formation an important cell signaling molecule adenosine via hydrolysis of adenosine monophosphate (AMP). This production of adenosine (along with other certain other signaling molecules) is the hallmark of ectonucleotidases involved in regulating the purinergic cell signaling. On this basis alone, APs can be considered as important modulators of purinergic cell signaling, however, as we have mentioned in our previous review [4], there is no unequivocal evidence available in the favor of this claim.

*Address correspondence to these authors at the Department of Chemistry, Forman Christian College (A Chartered University), Ferozepur Road, Lahore 54600, Pakistan; Tel:/Fax: ??????????; E-mail: [email protected]; and Centre for Advanced Drug Research, and Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad, Pakistan; Tel:/Fax: ?????????????; E-mails: [email protected], [email protected] 1389-5575/14 $58.00+.00

APs can be divided into two groups, the tissue nonspecific alkaline phosphatase (TNAP) and the tissue specific alkaline phosphatases. At least three different types of tissue specific alkaline phosphatase are known, the placental alkaline phosphatase (PLAP), intestinal alkaline phosphatase (IAP) and germ cell alkaline phosphatase (GCAP) [1, 5]. These APs have different gene expressions and exhibit different biochemical responses to specific inhibitors, which indicate differences in glycosylation patterns. Hence TNAP is efficiently inhibited by the well-known inhibitor levamisole but is not inhibited by L-phenylalanine-glycyl-glycine (PheGly-Gly), which is known to inhibit tissue specific alkaline phosphatases [6, 7]. The three tissue specific APs share 9098% sequence identity whereas the tissue non-specific alkaline phosphatase has only 50% sequence identity to that of tissue specific APs [8]. The distribution of APs in cells is ubiquitous and exact physiological functions of AP isozymes, other than the well understood TNAP, are rather elusive. An important role ofAPs in osteoblast and adipocyte differentiation and maturation pathway has been proven [9, 10]. AP is located on the surface of plasma membrane or secretory vesicles and is anchored to the cell surface via glycosylphosphatidyl inositol (GPI) linkages [11]. AP has been identified in a variety of adipocyte tissues and in preadipocytes [12]. Ali et al., [13] have demonstrated that AP is associated with the membrane surrounding intracellular lipid droplets and has been recognized to be crucial for adipogenesis. However the exact mechanism through which AP influences adipogenesis is not known. There is evidence of direct correlation between © 2014 Bentham Science Publishers

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Mini-Reviews in Medicinal Chemistry, 2014, Vol. 14, No. 13

AP activity and lipid accumulation, increased AP activity has been detected in parallel with the increase in fat accumulation in abdominal subcutaneous adipose tissue [14]. Based on this evidence, it was suggested that measurement of AP activity may in fact be a more rapid and sensitive technique for the quantification of intracellular lipid accumulation than any of the existing methods available [15]. It is important to note here that the AP responsible for regulating lipid accumulation and adipogenesis is of tissue non-specific type [16]. In humans, the body fat distribution was found to be linked with polymorphism in the genes encoding TNAP [17]. Role of TNAP in fat accumulation is further highlighted by studying TNAP knockout mice where severe lack of fat accumulation was observed [18]. TNAP was found to be elevated in inflamed colon of rat, moreover colonocytes and infiltrated leukocytes were found to be responsible for this increase in AP. Accordingly AP was suggested to be a relevant target for drug therapy [19, 20]. In 2009, Mizumori and co-workers [21] provided evidence of involvement of IAP in the regulation of alkaline pH of brush border membrane of duodenum. This is achieved via, what Mizumori describes as, an “ecto-purinergic pH regulatory system”. This regulatory loop consists of IAP, P2Y receptors (located on cell surface) and duodenal bicarbonate secretion stimulated by ATP (which acts on P2Y receptors), and ensures protection of mucosal membrane against acid induced injury. Inhibition of IAP (either due to inhibitor molecules or due to acidic environment) causes greater accumulation of ATP, which is no longer hydrolyzed by IAP, this ATP (a P2Y receptor agonist) stimulates P2Y receptors causing increased production of bicarbonate by the enterocytes, this bicarbonate is then transported to the luminal cavity where it neutralizes the acidic environment by reacting with H+ ions and producing H2O and CO2, this CO 2 is a substrate for carbonic anhydrase (CA) enzyme which again converts CO2 into bicarbonate ion with the release of H+ ions [21-23]. Role of TNAP in tissue mineralization has long been recognized by virtue of its high concentration in calcifying tissues such as bone and teeth [24]. However for proper mineralization to occur a template, collagen, is also needed to support precipitation of calcium phosphate [25]. AP aids in mineralization by providing phosphate pool, necessary for mineralization, as a result of its non-substrate specific phosphatase activity [26]. AP also has the ability to hydrolyze inorganic pyrophosphate (PPi), which is a potent inhibitor of mineralization [27]. Over-expression of TNAP causes disorders in normal bone formation process and is recognized as the main cause of hydroxyapatite deposition disorder (HADD, also known as calcific periarthritis or calcific tendinitis) which is characterized by unwanted deposition of basic calcium phosphate crystals in periarticular soft tissues especially tendons [28]. Ectopic calcification including vascular mineralization, calcification in renal failure and tumor calcification is also associated with increased levels of TNAP [26]. Therefore TNAP not only promotes mineralization, but together with ectonucleotide pyrophosphatase/ phosphodiesterase (NPP1/PC-1 which is responsible for maintaining enough levels of extracellular PPi) is also an important modulator of mineralization [29]. This inverse

Al-Rashida and Iqbal

concerted relationship of TNAP and NPP1 is crucial for normal bone formation [30]. AP isozymes (mainly PLAP) were found to be ectopically synthesized by a variety of tumors [31]. Cell lines derived from different cancers also retained the ability to produce these APs [32-35]. STRUCTURE OF ALKALINE PHOSPHATASES Alkaline phosphatases are homodimeric metal containing enzymes; the active site contains two zinc ions and one magnesium ion that are necessary for the catalytic function. For most part, the catalytic site is conserved in mammalian and bacterial APs, however mammalian APs have higher Km value are known to have optimum function at an alkaline pH. The crystal structure of human PLAP has been resolved and has greatly increased our understanding of structure and catalytic mechanism of APs [36]. Based on this crystal structure of PLAP and sequence alignment, structural models for other mammalian APs (IAP, GCAP and TNAP) were also built and compared [38]. These models confirm that most of the important residues involved in metal ion binding and catalytic mechanism are highly conserved in all mammalian APs. According to these models there is 98% identity between PLAP and GCAP isozymes. Between PLAP and IAP there exists 87% identity and 91% homology. TNAP is somewhat different and shares 57% identity and 74% homology with PLAP [38]. Important active site residues of PLAP involved in catalytic mechanism are Ser92, Asp42, His153, Ser155, Glu311, Asp316, His320, Asp357, His358, His360 and His432. Fig. (1) shows the catalytic active site of human PLAP [PDB-id, 1ZED] containing two Zn2+ and one Mg2+ ion along with their coordinated amino acid residues. One of the Zn2+ ions is directly coordinated to His320, His432 and Asp316, the fourth coordination site is occupied by a water molecule that can be replaced by a suitable substrate. The second Zn2+ ion is penta-coordinated and is directly coordinated to four amino acid residues His358, Asp357, Asp42 and Ser92, the fifth coordination site is occupied by water or a substrate molecule. The Mg2+ ion is hexa-coordinated and is directly coordinated to three amino acid residues, Asp42 (which is also coordinating the second Zn2+ ion), Ser155 and Glu311, and three water molecules which can be replaced upon substrate binding. A fourth metal ion, usually a Ca2+ ion is also present in all mammalian APs [39]. This feature, however, is not present in E. coli AP. The exact catalytic role or physiological function of this metal ion is still elusive but the presence of this fourth metal ion is crucial for normal functioning of the enzyme since mutations of the residues coordinating this fourth metal ion have been known to be linked with hypophosphatasia disorder characterized by defective bone mineralization [40]. INHIBITORS OF ALKALINE PHOSPHATASE In order to better understand the role of APs in health and in disease, it is imperative to develop small drug-like molecules that selectively inhibit APs. APs are uncompetitively inhibited by certain amino acids. The tissuespecific isozymes (PLAP, GCAP and IAP) are inhibited by L-phenylalanine but not by L-homoarginine, while reverse is true for the tissue non-specific TNAP [41]. Theophylline (1)

Inhibition of Alkaline Phosphatase: An Emerging New Drug Target


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Fig. (1). A view of catalytic active site of human PLAP (PDB-id, 1ZED). Figure has been created using Chimera [37].

[42-44] and (6S)-6-phenyl-2,3,5,6-tetrahydroimidazo[2,1b][1,3]thiazole commonly known as levamisole (2) [45, 46] are well known inhibitors of AP [19] (Fig. 2). One of the greatest drawbacks of the studies using levamisole to investigate role of AP in pathogenesis or in other regulatory mechanisms, is that levamisole has other known physiological effects as well, so it remains unclear whether the observed physiological effect is caused by levamisole mediated inhibition of AP or due to modulation of other pathways where levamisole has meddled [20]. New selective inhibitors of APs are therefore required in order to better elucidate the physiological roles of AP. A series of tetramisole [6-phenyl-2,3,5,6-tetrahydroimidazo [2,1-b][1,3]thiazole] derivatives containing methyl, nitro and halo substituents at the phenyl ring was synthesized and evaluated for their AP inhibitory activity [47]. Effect of replacement of phenyl group with naphthyl group was evaluated and was found to significantly increase the AP inhibitory activity [47]. Similarly selenium analog of tetramisole (selenotetramisole) (3) was also synthesized and tested for its AP inhibition activity [48]. The (-)-isomer of selenotetramisole was found to have a comparable inhibition profile to that of teramisole, however the other, (+)-isomer of selenotetramisole was found to be inactive [48]. A commonly used anticonvulsant barbiturate drug, Phenobarbital [5-ethyl-5phenyl-2,4,6(1H,3H,5H)-pyrimidinetrione] (4) was found to be an un-competitive inhibitor of all isozymes of AP over a wide concentration range of 20-400 mM [49]. Some

thiophenyl derivatives of levamisole were prepared by substituting the phenyl moiety with a thiophene moiety. The compounds exhibited potential to inhibit TNAP at pH 10.4. The IC50 values of different compound in the series ranged from an unimpressive 42 to >800 µM [50].

Fig. (2). Structures of compounds 1-5.

Much more potent TNAP inhibitory activity was observed for pyrazole derivatives, where the most active compound (5) had IC50 value of 0.005 µM [51]. Not only was compound (5) a potent inhibitor of TNAP, but also selective

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as it showed no inhibition against the placental PLAP isozyme. Detailed kinetic studies confirmed that compound (5) is a competitive inhibitor of TNAP, the possible mode of action for this inhibitor was rationalized using molecular docking studies employing homology built model of TNAP (since its crystal structure is not known) using sequence alignment and crystal structure of PLAP as a template. According to molecular docking studies there was an evidence of direct zinc binding that contributed to efficient inhibition. Three other selective and un-competitive TNAP inhibitors (6, 7, 8) were identified via high-throughput screening of chemical libraries [52]. These inhibitors did not inhibit the other isozymes PLAP and IAP either at physiological pH (7.5) or at alkaline pH (9.8). All three inhibitors were found to have better TNAP inhibition activities than standard inhibitor levamisole at pH 9.8 and 7.5 (Table 1). All compounds were able to significantly reduce calcification in cultured vascular smooth muscle cells (VSMCs). Moreover in ex vivo rat models, these compounds were able to inhibit build up of ectopic calcification by reducing the pyrophosphatase activity of TNAP. Inorganic pyrophosphatase (PPi) is a potent inhibitor of mineralization and is rapidly hydrolyzed by TNAP [53]. Therefore inhibiting the over expressed TNAP may be a desirable route for treating ectopic calcification including vascular calcification [54-57]. Another interesting route was adopted by O’Neill et al. in 2011 [58], they found that by increasing the amount of subcutaneously or intraperitoneally administered pyrophosphate in uremic rats, the calcium content in calcified aorta was reduced up to 70% with no significant side effects on bone formation [58]. Since patients with advanced stages of renal disease are more predisposed to the build-up of aortic calcification [56], this study warrants that further investigations into pyrophosphate therapy for treating vascular calcification in chronic renal disease patients may actually lead to promising results [59]. A series of benzo[b]thiophene derivatives was synthesized and screened against bovine IAP and TNAP [60]. Overall the compounds did not exhibit exceptional inhibition activities, the percentage inhibitions calculated at 0.4 mM concentration of inhibitor were in the range 111% to 44% against TNAP; and against IAP were 129% to 43% when determined against 0.1 mM concentration of inhibitor. Some compounds indicated a slight preference of IAP over TNAP but with unimpressive inhibition constants. Ki values of only two compounds 6-(1-benzothiophen-3-yl)-5,6-dihydroimidazo [2,1-b][1,3]thiazole (9), and its tetrahydro isomer (10), were calculated and found to be 85 ± 6 and 135 ± 3 respectively against TNAP (Fig. 3).

Fig. (3). Structures of compounds 9 and 10.


Table 1.

TNAP inhibition activities of compounds 6-8 at pH 9.8 and 7.5 respectively. TNAP Compound

Ki (µM) ± SEM pH 9.8

Ki (µM) ± SEM pH 7.5

5.6±1.6

6.4±1.6

5.6±0.7

33±5.8

6.5±1.4

2.8±0.4

N N NH O S

(6) O N

S

N

NH

O

NH

(7)

NH N N

N S

N N

NH

(8)

A series of arylsulfonamide derivatives (11-24) was found to be potent inhibitors of TNAP [61] (Table 2). The structural elements found essential for potent TNAP inhibition were the presence of 3-quinoline or 3-pyridine groups on sulfonamide nitrogen atom, where quinoline based derivatives were in general more active than corresponding pyridine analogs. Another important structural element identified was the presence of alkoxy group on the ortho position of the aryl ring bearing sulfonamide group. Other aryl substituents were found to have less dramatic effects on TNAP inhibition activity. Most of these compounds were found to be selective TNAP inhibitors showing little or no inhibition activity towards related isozymes PLAP and IAP. Compound (9) was the most selective showing IC50 values of >100 against both PLAP and IAP. Three main scaffolds, the bisarylsulfonamides, pyrazole and triazole were identified as potent TNAP inhibitors in high throughput screening assays [62]. For bisarylsulfonamide scaffold, the hits identified are mainly based on the previously reported compounds which are summarized in Table 2. Most of these bisarylsulfonamides did not exhibit any inhibition activity against PLAP or IAP. Interestingly when the sulfonamide linker group (SO2NH) was replaced with CH2NH, NHCH2 or even CONH all compounds lost their TNAP inhibition activity (Fig. 4). Most promising and active TNAP inhibitors (25-27) based on pyrazole and triazole scaffold are given below in Table 3. The pyrazole based compounds indicated selectivity for TNAP over PLAP and IAP, whereas the triazole based compound did not exhibit any marked preference for any isozyme. The compounds in table below are important lead


Table 2.


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TNAP inhibition activities of compounds 11-24. Compound

TNAP IC 50 µM

Compound

O

O

TNAP IC 50 µM

O

O S NH O

Cl

N

O S NH O O

0.19

N

1.13

(18)

(11) F

O O S NH O O

N

O S NH O O

1.06

(12)

N

1.29

(19)

Cl

O

O S NH O O

N

O S NH O O

0.51

(13)

N

1.49

(20)

Cl

O

O S NH O O

N

Br O S NH O O

0.54

N

1.55

(21)

(14) Cl O S NH O O

N O S NH O O

0.69

N

1.68

(22)

(15) Br O S NH O O

N

O S NH O O

1.13

0.12

(23)

(16)

O S NH O O

N

O 2N

N

0.65

(17)

molecules that can be optimized in pursuit of better selectivity and inhibition profiles. The results for these compounds are based on luminescent assay [62]. Other than the ubiquitously present amino acids, there is a significant lack of known IAP inhibitors especially selective IAP inhibitors. Two compounds (28) and (29) were identified as active IAP inhibitors (IC50 µM; 0.061±0.001 and 0.15±0.02 respectively) [63]. Two more related compounds (30) and (31) were also identified as IAP

O S NH O O

N

0.74

(24)

inhibitors (IC50 µM; 0.92±0.03 and 1.32±0.20 respectively) [64]. Structures of compounds 28-31 along with their IC50 values are given in Table 4. None of these compounds were tested against any other isozyme of AP so their selectivity (if any) remains unknown. It is also important to note that all these compounds were assayed using colorimetric methods employing p-nitrophenyl acetate hydrolysis; the success of luminescent based assay over colorimetric assay is well established and has been thoroughly investigated [62].

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Fig. (4). Replacement of sulfonamide linker group (SO2NH) with CH2NH, NHCH2 and CONH ceases TNAP inhibition activity of bisarylsulfonamides. Table 3.

TNAP inhibition activities of compounds 25-27. Compound

TNAP IC50 µM

PLAP IC50 µM

IAP IC50 µM

0.19

3.1

0.54

4.3

>100

>100

0.74

>100

44

O N O

N N N H

(25)

HO

N NH

(26)

Cl HO O

N NH

(27)

Some IAP selective triazole based inhibitors (32-38) were identified in a high throughput screening assay [62]. However all inhibitors did not exhibit exceptional preference towards IAP, structures of some of the potent IAP inhibitors along with their IC50 values obtained from a luminescent based assay are given in Table 5. Using an ultrahigh throughput chemiluminescent assay compound libraries were screened for AP inhibition activities. This search identified compounds selective for murine IAP inhibition [65]. Recently a new sulfonamide containing compound (39) (Fig. 5) was identified (in a high through put assay) as a potent and selective inhibitor of murine IAP [66].

Fig. (5). Structure of compound 39, a selective murine IAP inhibitor.


Table 4.

IAP inhibition activities of compounds 28-31. IAP IC50 ± SEM (µM)

Compound O

N N

Br

0.061±0.001

S

N N


inhibitory activities [75]. Peroxo vanadium and peroxo tungstate complexes with amino acid ligands were synthesized and found to be active inhibitors of IAP from rabbit intestine. Kinetic studies indicated mixed type inhibition by these compounds [76]. Similar complexes with cystine [77] and dipeptides (glycyl-leucine or glycyl-glycine) as ancilliary ligands were also found to be active inhibitors of AP [78]. Table 5.

TNAP and IAP inhibition activities of compounds 32-38.

(28) O

Compound O

N N

TNAP IC50 ± SEM (µM)

IAP IC50 ± SEM (µM)

0.80

0.12

1.57

1.10

27.9

0.72

>100

1.75

30.6

4.75

10.5

1.77

4.68

0.41

0.15±0.02

S

O

N N

N

(29)

N

O

Cl

N N H

(32)

N

F

N N

S

O

0.92±0.03 N

N N

N

O

N N H

(30)

(33)

Cl Cl

O N

N

N N

1.32±0.20

O

N N N H

S

(34)

N N

(31)

O

A series of novel chromones containing sulfonamides (40-47) was synthesized and evaluated as inhibitors of TNAP and IAP [67]. Most of the compounds in the series were selective IAP inhibitors. Detailed structure activity relationship studies were carried out that attempted to highlight the structural elements that could be modulated for the design of more potent and selective inhibitors of AP. Some of the more promising inhibitors from this series are given in Table 6. POLYOXOMETALATES AND BASED INHIBITORS OF AP

OTHER

N O

N N N H

(35) O N O

N N N H

(36)

METAL

Sodium vanadate has long been known to inhibit AP in a reversible manner [68, 69]. Moreover oxyanions such as tungstate, molybdate [70, 71] and vanadate [72] competitively inhibit AP. The exact mechanism through which polyoxometalates achieve AP inhibition remains unknown, however, it has been speculated that it is probably due to fact their penta- or hexa-coordinate complexes can mimic phosphate analogs. This hypothesis seems to be in correlation with the fact that vanadate complexes have insulin mimetic activities [73, 74]. Vanadium (IV) metal complexes with ferulic acid (4-hydroxy-3-methoxy cinnamic acid) and cinnamic acid were found to have similar IAP

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O N O

N N

N N H

(37) O N O

N N N H

(38)

O

8


Table 6.

TNAP and IAP inhibition activities of compounds 40-47. TNAP Ki ± SEM (µM)

Compound O

O

O

HN


(Table 6) Condt….

IAP Ki ± SEM (µM)

Compound O

O

O

HN

TNAP Ki ± SEM (µM)

IAP Ki ± SEM (µM)

21±7.8

0.01±0.01

22±5.9

0.41±0.06

F O S NH2 O

6.8±1.0

90.6±8.9

O S N S NH N O

(40)

(46)

O

O

O

HN

O

O

O

HN

Br

41±8.3 O S O NH2

s

O S NH O

33.5±8.0

N

(47)

(41) O

O

O

HN

F O S NH2 O

220±37

1.43±0.04

18±2.2

17.1±0.02

48±13

0.41±0.02

(42) O

O

O

HN

F

O S O NH2

(43) O

O

O

HN

Br O S NH2 O

(44) O

O

O

HN

Br

44±6.7

8.7±0.09

27.3±5.8

0.049±0.007

O S O NH2

O

H N

O HN

S O O

(45)

A series of POMs was synthesized and found to possess good AP inhibitory activity against both TNAP and IAP. Two complexes in the series, Na10[H2W12O42]·27H2O (Ki = 313±7 nM) and Na33[H7P8W48O184]·92H2O (Ki = 135±10 nM) were most active inhibitors of IAP and TNAP respectively [79]. Nanoassemblies of POMs using chitosan, a naturally occurring biopolymer, as a template were synthesized and were found to be active inhibitors of IAP and TNAP. The biopolymer derived nanoassemblies were more potent inhibitors as compared to the parent POM [80]. A major disadvantage of use of these polyoxometalate compounds is their hydrolytic instability which limits their usefulness as potential therapeutic agents. A step forward, and certainly in the right direction, has been the synthesis of polymer bound POMs. Molybdenum (VI ) and vanadium (V) polymer bound peroxo complexes have been synthesized in water soluble polymers such as poly(acrylate), poly (methacrylate), poly(acrylamide), poly(styrene sulfonate), and poly(styrene sulfonate-co-maleate) [81]. This strategy has the advantage of rendering stability and resistance towards hydrolysis to the POMs. Moreover, such polymer bound POMs were even resistant to enzyme hydrolysis by catalases. The polymer bound complexes exhibited noncompetitive type inhibition, in contrast to the mixed type inhibition exhibited by corresponding free POMs. In conclusion APs are important enzymes with multifaceted physiological roles. The role of tissue non-specific alkaline phosphatase (TNAP) in regulation of mineralization and bone formation is firmly established. Modulation in normal levels of TNAP is associated with disordered bone formation such as hypophosphatasia, hydroxyapetite deposition disorder, vascular and arterial calcification. TNAP, therefore, has been identified as an important drug target for the potential treatment of such diseases. Selective inhibitors of APs, thus, may not only have therapeutic potential, they are also required to elucidate in depth the physiological and pathophysiological roles of some of the less commonly studied AP isozymes (mainly IAP, followed by PLAP and GCAP).



LIST OF ABBREVIATIONS AP/s

= Alkaline phosphatase/s

E-NTPDase/s = ecto-nucleoside triphosphate diphosphohydrolase/s E-NPP/s

= ecto-nucleotide pyrophosphatase/phosphodiesterases

e5ʹNT

= ecto-5ʹ-nucleotidase

TNAP

= Tissue non-specific alkaline phosphatase

GCAP

= Germ cell alkaline phosphatase

IAP

= Intestinal alkaline phosphatase

PLAP

= Placental alkaline phosphatase

Pi

= Inorganic phosphate

PPi

= Inorganic pyrophosphate

GPI

= Glycosylphosphatidyl inositol

CA

= Carbonic anhydrase

HADD

= Hydroxyapatite deposition disorder

AMP

= Adenosine monophosphate

ATP

= Adenosine triphosphate

PC-1

= Plasma cell membrane glycoprotein-1

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Received: ?????????, 2014

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Revised: ?????????, 2014

Accepted: ?????????, 2014