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Synthesis and evaluation of novel purple acid phosphatase inhibitors.

ABSTRACT: Transgenic studies in animals have demonstrated a direct association between the level of expression of purple acid phosphatase (PAP; also known as tartrate-resistant acid phosphatase) and the progression of osteoporosis. Consequently, PAP has emerged as a promising target for the development of novel therapeutic agents to treat this debilitating disorder. PAPs are binuclear hydrolases that catalyse the hydrolysis of phosphorylated substrates under acidic to neutral conditions. A series of phenyltriazole carboxylic acids, prepared by the reactions of azide derivatives with propiolic acid through copper(i)-catalysed azide-alkyne cycloaddition click reactions, has been assessed for their inhibitory effect on the catalytic activity of pig and red kidney bean PAPs. The binding mode of most of these compounds is purely uncompetitive with K iuc values as low as ?23 ?M for the mammalian enzyme. Molecular modelling has been used to examine the binding modes of these triazole compounds in the presence of a substrate in the active site of the enzyme in order to rationalise their activities and to design more potent and specific derivatives.

SUBMITTER: Hussein WM

PROVIDER: S-EPMC6350629 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Synthesis and evaluation of novel purple acid phosphatase inhibitors.

Hussein Waleed M WM Feder Daniel D Schenk Gerhard G Schenk Gerhard G Guddat Luke W LW McGeary Ross P RP

MedChemComm 20181113 1

Transgenic studies in animals have demonstrated a direct association between the level of expression of purple acid phosphatase (PAP; also known as tartrate-resistant acid phosphatase) and the progression of osteoporosis. Consequently, PAP has emerged as a promising target for the development of novel therapeutic agents to treat this debilitating disorder. PAPs are binuclear hydrolases that catalyse the hydrolysis of phosphorylated substrates under acidic to neutral conditions. A series of pheny ...[more]

PMID: 30774855

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Project description:The tartrate-resistant acid phosphatase (TRAP) of rat osteoclasts has been shown to exhibit high (85-94%) identity at the amino acid sequence level with the purple acid phosphatase (PAP) from bovine spleen and with pig uteroferrin. These iron-containing purple enzymes contain a binuclear iron centre, with a tyrosinate-to-Fe(III) charge-transfer transition responsible for the purple colour. In the present study, production of rat osteoclast TRAP could be achieved at a level of 4.3 mg/litre of medium using a baculovirus expression system. The enzyme was purified to apparent homogeneity using a combination of cation-exchange, hydrophobic-interaction, lectin-affinity and gel-permeation chromatography steps. The protein as isolated had a purple colour, a specific activity of 428 units/mg of protein and consisted of the single-chain form of molecular mass 34 kDa, with only trace amounts of proteolytically derived subunits. The recombinant enzyme had the ability to dephosphorylate bone matrix phosphoproteins, as previously shown for bone TRAP. Light absorption spectroscopy of the isolated purple enzyme showed a lambda max at 544 nm, which upon reduction with ascorbic acid changed to 515 nm, concomitant with the transition to a pink colour. EPR spectroscopic analysis of the reduced enzyme at 3.6 K revealed a typical mu-hydr(oxo)-bridged mixed-valent Fe(II)Fe(III) signal with g-values at 1.96, 1.74 and 1.60, proving that recombinant rat TRAP belongs to the family of PAPs. To validate the use of recombinant PAP in substituting for the rat bone counterpart in functional studies, various comparative studies were carried out. The enzyme isolated from bone exhibited a lower K(m) for p-nitrophenyl phosphate and was slightly more sensitive to PAP inhibitors such as molybdate, tungstate, arsenate and phosphate. In contrast with the recombinant enzyme, TRAP from bone was isolated predominantly as the proteolytically cleaved, two-subunit, form. Both the recombinant enzyme and rat bone TRAP were shown to be substituted with N-linked oligosaccharides. A slightly higher apparent molecular mass of the monomeric form and N-terminal chain of bone TRAP compared with the recombinant enzyme could not be accounted for by differential N-glycosylation. Despite differences in specific post-translational modifications, the recombinant PAP should be useful in future studies on the properties and regulation of the mammalian PAP enzyme.

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Synthesis and evaluation of novel purple acid phosphatase inhibitors.

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Synthesis and evaluation of novel purple acid phosphatase inhibitors.

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