Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

Elliott, Tristan W; Mitić, Nataša; Gahan, Lawrence R.; Guddat, Luke W.; Schenk, Gerhard

doi:10.1590/s0103-50532006000800011

Cited by 34 publications

(42 citation statements)

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“…28 Small anions, including fluoride and tetrahedral oxoanions such as phosphate, vanadate and tungstate are known to inhibit PAP. 1,[29][30][31] Myers and coworkers 32 have described a number of simple phosphonates with pendant metal-binding groups, such as thiol, phosphonate and carboxylate, and these have shown inhibitory activity against rkbPAP, in the range 80-3000 lM. Phosphotyrosyl-containing tripeptides are excellent substrates for PAP.…”

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confidence: 99%

“…33 The validity of using rkbPAP and pPAP as models of the human enzyme is supported by a number of factors including (i) the high degree of structural conservation in their substrate-binding pockets, 9,10,[14][15][16] (ii) their conserved reaction mechanism(s), 1 and (iii) the similarity of inhibition constants for a range of inhibitors reported for several animal (including human) and plant PAPs (e.g., the fluoride inhibition constants of rkbPAP, pPAP and hPAP are 170, 120 and 170 lM, respectively). 1,11,17,[28][29][30][31] Furthermore, mammalian and rkbPAPs have similar catalytic efficiencies and catalyse the hydrolysis of similar substrates. 1,11,30 Computer modelling was used to identify new compounds that might act as inhibitors of PAPs.…”

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confidence: 99%

“…The inhibitory effects of the new compounds were tested with both rkbPAP and pPAP at pH 6.2 and 4.9, the respective pH optima 28,31 of the two enzymes (Tables 1 and 2). Hydroxylation of the benzylic carbon of 1, to give compound 2, had very little effect on the binding affinity.…”

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Inhibition of purple acid phosphatase with α-alkoxynaphthylmethylphosphonic acids

McGeary

Vella

Mak

et al. 2009

Bioorganic & Medicinal Chemistry Letters

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a b s t r a c tPurple acid phosphatases (PAPs) are binuclear hydrolases that catalyse the hydrolysis of a range of phosphorylated substrates. Human PAP is a major histochemical marker for the diagnosis of osteoporosis. In patients suffering from this disorder, PAP activity contributes to increased bone resorption and, therefore, human PAP is a key target for the development of anti-osteoporotic drugs. This manuscript describes the design and synthesis of derivatives of 1-naphthylmethylphosphonic acids as inhibitors of PAP. The K i values of these compounds are as low as 4 lM, the lowest reported to date for a PAP inhibitor.

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Inhibition of purple acid phosphatase with α-alkoxynaphthylmethylphosphonic acids

McGeary

Vella

Mak

et al. 2009

Bioorganic & Medicinal Chemistry Letters

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“…The numbers between the motif blocks indicate the numbers of amino acid residues between each motif, and 'length' shows the total number of amino acid residues of each PAP. Plants 35 and Plants 55 are small and large forms of plant PAPs, respectively. b Incomplete determination by amino acid sequencing.…”

Section: Current Osteoporosis Treatmentsmentioning

confidence: 99%

“…81 The overall catalytic step involves a direct transfer of the phosphate moiety of the substrate to a nucleophilic solvent molecule that is coordinated to a metal ion. 55,65,[72][73][76][77]107 The first step involves the binding of the substrate to PAP to form a pre-catalytic complex. There is no experimental data of this structure currently available, however the crystal structure of red kidney bean PAP (rkbPAP)-sulfate complex solved by Schenk and co-workers at 2.4 Å (Figure 6) shows that instead of binding directly to the metal ions, the sulfate is positioned in the second coordination sphere, and is stabilised via hydrogen bonds with the µ-hydroxide, His202, His295, His296 and Asn201 (residue numbers refer to the sequence of rkbPAP).…”

Section: Mechanism Of Actionmentioning

confidence: 99%

Design, synthesis and testing of purple acid phosphatase inhibitors for the development of anti-osteoporotic drugs

Pahmi¹

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Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have been isolated from various mammals, plants, fungi and bacteria. They catalyse phosphoric ester hydrolysis under acidic to neutral conditions by utilising two closely spaced heterovalent metal ions in their active sites. In mammals, PAP activity is associated with bone resorption, which can lead to bone diseases such as osteoporosis. For instance, osteoporosis patients are observed to have elevated PAP serum levels.Furthermore, research has shown that transgenic mice that over-expressed the PAP gene developed mild osteoporosis. These observations therefore make human PAP an attractive target to develop anti-osteoporotic drugs.This project employed the strategy of using a substrate analogue, where the lead compound (as shown below) with K i value of 8 µM against pig PAP was chosen as the basis in this rational drug design. Therefore acyl derivatives of α-aminoarylmethylphosphonic acids (as shown below) were designed as potential new potent PAP inhibitors. Docking studies suggested that the derivatives would have high binding affinity due to the phosphonate moiety (substrate mimic) binding to the binuclear metal centre, which are as intended. Furthermore, the long acyl chains make favourable interactions with the long hydrophobic groove adjacent to the dimetal centre, and depending on the size and substitutions on the aromatic ring, the ring might occupy the space in any of the small pockets in the vicinity of the active site; all of which contribute to the predicted high binding affinities of the derivatives to the enzyme.The lead compound: (naphthalene-1-yl(tetradecanamido)methyl)phosphonic acidR 2 = Me(CH 2 ) m -, Me(CH 2 ) n OCH 2 -; m = 8, 10, 12, 14, 16; n = 7, 15, 17. Acyl derivatives of α-(amino(aryl)methyl)phosphonic acids IIIThe derivatives were synthesised in three or four steps method in good yields and then tested as pig PAP inhibitors. The kinetic studies in this project were performed using newly extracted and purified pig PAP. The extraction and purification of pig PAP from the allantoic fluid of a pregnant pig was successfully carried out using fast protein liquid chromatography (FPLC) by adapting a well-established protocol. Kinetic analysis showed that the derivatives have high inhibition potencies against pig PAP with K i values in the low micromolar to nanomolar range. The most potent pig PAP inhibitor within this series was the octadecyl-derivative of α-(phenyl(amido)methyl)phosphonic acid with K i value of 168 nM. This is the most potent pig PAP inhibitor to date.Screening tests on a hundred other compounds were also performed to identify new possible alternative PAP inhibitors. Most of the compounds have metal-binding moieties as they were developed as inhibitors of a metallo-β-lactamase, which is also a binuclear metallohydrolase.Therefore it was thought that they might have inhibition properties against PAPs. However, they were found to have little or no inhibition against pig PAP. Nonetheless, these results ...

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The Binding Mode of an ADP Analogue to a Metallohydrolase Mimics the Likely Transition State

et al. 2019

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Purple acid phosphatases( PAPs) are members of the large family of metallohydrolases, ag roup of enzymes that perform aw ide range of biological functions, while employing ah ighly conserved catalytic mechanism. PAPs are found in plants,a nimals and fungi;i nh umans they play an important role in bone turnovera nd are thus of interest for developingt reatments for osteoporosis. The majority of metallohydrolases use am etalbound hydroxide to initiate catalysis, which leads to the formation of ap roposed five-coordinate oxyphosphorane species in the transition state. In this work, we crystallized PAPf rom red kidney beans (rkbPAP) in the presence of both adenosine and vanadate. The in crystallo-formed vanadate analogue of ADP provides detailed insight into the binding mode of aP AP substrate, captured in as tructure that mimics the putative fivecoordinate transition state. Our observations not only provide unprecedented insightinto the mechanism of metallohydrolases, but might also guide the structure-based design of inhibitors for application in the treatment of severalhuman illnesses.Metallohydrolases form al arge familyo fm ostly bimetallic enzymes that use metal ionst oactivate both the nucleophile and scissile bond of various phosphate or amide substrates. [1][2][3][4] Examples include the antibiotic-degrading metallo-b-lactamases, pesticide-decontaminating organophosphate hydrolases, and purple acid phosphatases (PAPs). [5][6][7] PAPs, the only known metallohydrolases that require ah eterovalent Fe III M II center (with M = Fe, Zn or Mn) for catalytic activity, have been characterized from various mammals, plants and fungi. [2,5] Mostp lant PAPs use either Zn II or Mn II ,w hereas their mammalianc ounterparts employ ar edox-activei ron ( Figure 1). [8,9] The reported substrates for PAPs include adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate( ADP), phosphotyrosine and pyrophosphate. [5,10] The synthetic substrate para-nitrophenyl phosphate (pNPP) is frequently used for functionalstudies. [2,5] The biological functions of PAPs are diverse and dependent on the organism. For mammals, experiments with transgenic mice have demonstrated that PAPp lays an important role in bone metabolism. [11,12] In plants, PAPs playamajor role in the acquisition of phosphorus, especially if there is al imited supplyo fp hosphate. [13,14] Due to their diverse roles, PAPs have attracted attention either as targetsf or new treatments of osteoporosis or for applicationsi na griculture to aid nutrient uptake by crops. [11,15] PAPs operate optimally in the pH range between5 .0 and 6.5, and are characterized by ad istinct purple color due to ac harge-transfer transition between an active-site tyrosine ligand and an Fe III ion. [16,17] The crystal structures of several mammalian and plant PAPs have demonstrated that, despite the difference in the metal ion selectivity and the limited degree of sequence similarity,t heir actives ite geometries are highly conserved (Figure1). [18][19][20][21][22][23] Consequently,t he proposed mod...

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Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

Cited by 34 publications

References 40 publications

Inhibition of purple acid phosphatase with α-alkoxynaphthylmethylphosphonic acids

Inhibition of purple acid phosphatase with α-alkoxynaphthylmethylphosphonic acids

Design, synthesis and testing of purple acid phosphatase inhibitors for the development of anti-osteoporotic drugs

The Binding Mode of an ADP Analogue to a Metallohydrolase Mimics the Likely Transition State

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