Tissue-nonspecific Alkaline Phosphatase Inhibitor Research Articles

Identification and Characterization of Novel Tissue-Nonspecific Alkaline Phosphatase Inhibitors with Diverse Modes of Action

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitous enzyme expressed at high levels in bone, liver, and kidney. It appears involved in dephosphorylation of numerous phosphate monoesters, but only 2 of them, pyrophosphate and pyridoxal phosphate, have yet been unequivocally documented. Discovery and characterization of other substrates could be considerably facilitated if specific and potent modulators of TNAP activity with various modes of action were available. Here, the authors describe in detail a high-throughput screening campaign to identify inhibitors of TNAP, performed within the Molecular Library Screening Center Network (MLSCN). A novel homogeneous luminescent TNAP assay was developed and optimized with respect to the enzyme and substrate concentrations, enabling identification of a large number of compounds overlooked by a conventional colorimetric assay. Several new chemical series were identified from screening the Molecular Libraries Small Molecule Repository (MLSMR) collection and demonstrated to have diverse selectivity and mode of inhibition profiles. The nanomolar potency of some of these scaffolds surpasses currently known inhibitors. This article provides an example of a success where the Roadmap Initiative collaborative model, sponsored by the National Institutes of Health, brought together a deep knowledge of target biology from a principal investigator's laboratory, a well-designed compound collection from the MLSMR, and an industrial-level screening facility and staff at the MLSCN center to identify pharmacologically active compounds, with outstanding selectivity data from a panel of more than 200 publicly accessible assays, through a high-throughput screen.

Lansoprazole is an uncompetitive inhibitor of tissue-nonspecific alkaline phosphatase.

Lansoprazole, a known H(+)/K(+)-ATPase inhibitor, is currently used as a therapeutical option for the initial treatment of gastroesophageal reflux disease. Recently, lansoprazole has been found to be an inhibitor of cytosolic PHOSPHO1 (a phosphatase which hydrolyses phosphocholine and phosphoethanolamine), providing a possible therapeutical target to cure pathological mineralization. Since PHOSPHO1 is present inside matrix vesicles, we tested the effect of lansoprazole on matrix vesicles containing several key enzymes for the mineralization process including tissue-nonspecific alkaline phosphatase. We found that lansoprazole can inhibit in an uncompetitive manner tissue-nonspecific alkaline phosphatase. A K(i) value of 1.74 +/- 0.12 mM has been determined for the inhibition of tissue-nonspecific alkaline phosphatase by lansoprazole. Lansoprazole, currently used for treating gastroesophageal disease, by inhibiting PHOSPHO1 and tissue-nonspecific alkaline phosphatase could prevent hydroxyapatite-deposition disease and could serve as an adjunct treatment for osteoarthritis.

Design and synthesis of pyrazole derivatives as potent and selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP)

Tissue-nonspecific alkaline phosphatase (TNAP) plays a central role in regulating extracellular matrix calcification during bone formation and growth. High-throughput screening (HTS) for small molecule TNAP inhibitors led to the identification of hits in the sub-micromolar potency range. We report the design, synthesis and in vitro evaluation of a series of pyrazole derivatives of a screening hit which are potent TNAP inhibitors exhibiting IC 50 values as low as 5 nM. A representative of the series was characterized in kinetic studies and determined to have a mode of inhibition not previously observed for TNAP inhibitors.

The role of tissue-nonspecific alkaline phosphatase in the phosphate-induced activation of alkaline phosphatase and mineralization in SaOS-2 human osteoblast-like cells

Tissue-nonspecific alkaline phosphatase (TNAP) plays a key role in mineralization by degrading inorganic pyrophosphate and providing free inorganic phosphate. We have previously reported that TNAP is induced by beta-glycerophosphate and NaH(2)PO(4) in short-term cultures of SaOS-2 human osteoblast-like cells and that PHEX (phosphate-regulating gene with homologies to endopeptidase on the X chromosome) mRNA is also induced after TNAP induction. In the present study, we have investigated the effects of levamisole, a TNAP inhibitor, and phosphonoformic acid (PFA), a type III sodium-phosphate cotransporter inhibitor, on the phosphate-induced expression of TNAP and mineralization. Levamisole inhibited beta-glycerophosphate-induced mineralization, TNAP and PHEX expression, and the increase in enzymatic activity of NPP1 (5'-nucleotide pyrophosphatase phosphodiesterase 1), but did not inhibit NaH(2)PO(4)-induced mineralization. PFA completely inhibited NaH(2)PO(4)-induced mineralization and NPP1 enzymatic activation, and partly inhibited beta-glycerophosphate-induced mineralization, but did not affect the increase in TNAP activity. These results suggest that phosphate derived from TNAP-induced hydrolysis of beta-glycerophosphate yields signals that induce TNAP expression and mineralization, and that PHEX expression may be linked to TNAP expression. However, luciferase assays failed to detect any transcriptional activation of the promoter region of the human TNAP gene by beta-glycerophosphate or NaH(2)PO(4), suggesting that the effects of these phosphates may be indirect.

Tissue non-specific alkaline phosphatase and tibial size in mice of a F2 segregating population

Atherosclerotic plaque calcification varies from early, diffuse microcalcifications to a bone-like tissue formed by endochondral ossification. Recently, a paradigm has emerged suggesting that if the bone metaplasia stabilizes the plaques, microcalcifications are harmful. Tissue-nonspecific alkaline phosphatase (TNAP), an ectoenzyme necessary for mineralization by its ability to hydrolyze inorganic pyrophosphate (PPi), is stimulated by inflammation in vascular smooth muscle cells (VSMCs). Our objective was to determine the role of TNAP in trans-differentiation of VSMCs and calcification. In rodent MOVAS and A7R5 VSMCs, addition of exogenous alkaline phosphatase (AP) or TNAP overexpression was sufficient to stimulate the expression of several chondrocyte markers and induce mineralization. Addition of exogenous AP to human mesenchymal stem cells cultured in pellets also stimulated chondrogenesis. Moreover, TNAP inhibition with levamisole in mouse primary chondrocytes dropped mineralization as well as the expression of chondrocyte markers. VSMCs trans-differentiated into chondrocyte-like cells, as well as primary chondrocytes, used TNAP to hydrolyze PPi, and PPi provoked the same effects as TNAP inhibition in primary chondrocytes. Interestingly, apatite crystals, associated or not to collagen, mimicked the effects of TNAP on VSMC trans-differentiation. AP and apatite crystals increased the expression of BMP-2 in VSMCs, and TNAP inhibition reduced BMP-2 levels in chondrocytes. Finally, the BMP-2 inhibitor noggin blocked the rise in aggrecan induced by AP in VSMCs, suggesting that TNAP induction in VSMCs triggers calcification, which stimulates chondrogenesis through BMP-2. Endochondral ossification in atherosclerotic plaques may therefore be induced by crystals, probably to confer stability to plaques with microcalcifications.

Pharmacology and Therapeutics

<h3>Background</h3> Inhibiting ecto-5’-nucleotidase (CD73) to reduce immunosuppressive adenosine in the tumor microenvironments is an anti-tumor strategy currently explored in clinical trials. Measuring soluble CD73 (sCD73) activity in plasma to evaluate pharmacodynamics of CD73 inhibitors is appealing. Quantifying phosphate in plasma after adding exogenous adenosine monophosphate (AMP) can be used to determine sCD73 activity.¹ Maintaining high plasma concentration to prevent dilution of endogenous sCD73 when quantifying its activity is desirable. High protein concentrations in plasma, however, can affect accurate phosphate quantitation. By precipitating plasma proteins prior to phosphate quantitation, we developed and qualified a method to determine sCD73 activity in 95% plasma. <h3>Methods</h3> Platelet poor heparinized plasma (PPP), AMP, tissue nonspecific alkaline phosphatase inhibitor (TNAPi), recombinant CD73, malachite green, adenosine 5’- (α, β-methylene) diphosphate (APCP), and recombinant alkaline phosphatase (ALP) were procured commercially. sCD73 concentrations were measured by ELISA and total protein concentration was quantified by BCA. sCD73 activity was measured by combining PPP, TNAPi, and AMP at 37°C. Reactions were terminated with trichloroacetic acid (TCA) at various timepoints to generate a kinetic readout. After protein precipitation, phosphate concentrations were measured by malachite green and enzymatic rates calculated as change in free phosphate concentration per minute. <h3>Results</h3> Incubating TCA-terminated reaction mixtures at 4°C for ≥ 3 hours reduced protein in supernatants to below lower limits of quantitation and eliminated interference in phosphate detection. Plasma sCD73 activity was dependent on AMP concentrations (Km = 612 μM), proportional to sCD73 in the sample and could be fully inhibited by APCP. 500 μM TNAPi, an inhibitor of non-CD73 AMPase activity, fully blocked 670 IU/L of recombinant human ALP activity. sCD73 activity in PPP from colorectal carcinoma (CRC) patients was higher (p= 0.0028) than in healthy volunteers (HV). sCD73 activity in some individuals with gastric cancer (GC), non-small cell lung cancer (NSCLC), and triple-negative breast cancer (TNBC) were also numerically higher than in healthy volunteers (figure 1a). sCD73 activity was correlated to sCD73 concentrations in these samples (figure 1b). The method was qualified for use in clinical studies (table 1). Plasma sCD73 activity assay characterization and fit-for-purpose qualification. Abbreviations: LOD, limit of detection; LLOQ, lower limit of quantitation; ULOQ, upper limit of quantitation; APCP, adenosine 5’- (α, β-methylene) diphosphate. <h3>Conclusions</h3> A method to quantify sCD73 activity in 95% plasma to evaluate pharmacodynamics of CD73 inhibitors in clinical samples was developed and qualified. Plasma sCD73 activity was dependent on AMP concentration and inhibited by APCP. Plasma sCD73 activity was significantly elevated in CRC patients and selected patients with GC, NSCLC, and TNBC and was proportional to sCD73 concentration. <h3>Reference</h3> Morello S, Capone M, Sorrentino C, Giannarelli D, Madonna G, Mallardo D, Grimaldi AM, Pinto A, Ascierto PA. Soluble CD73 as biomarker in patients with metastatic melanoma patients treated with nivolumab. <i>J Transl Med</i> 2017 Dec 4;<b>15</b>(1):244. doi: 10.1186/s12967-017-1348-8. PMID: 29202855; PMCID: PMC5716054.