Identification and characterization of novel tissue-nonspecific alkaline phosphatase inhibitors with diverse modes of action

Abstract

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.

Figures

Figure 1

HTS data for the luminescence-based TNAP assay. MLSMR compound collection was screened at 20 uM compound concentration in the assay. The actual experimental data from screening 191 assay plates on seven different days is displayed in panel A without further modifications. Compounds inhibition calculated according to Eq. 1 is summarized in panel B in a histogram format. Tested compounds and control wells are depicted in blue and red color, respectively. Graphs are prepared using Spotfire software (TIBCO).

Figure 2

Confirmation of primary TNAP hits. Cherry-picked hits were tested at 20 uM concentration in the luminescent (A and B) and colorimetric (C and D) assays. The concentration of DEA was 100 mM (A) or 1M (B, C, and D). Activity of the enzyme in the colorimetric assays was measured using absorbance of either p-nitrophenyl (C) or malachite green-phosphomolybdate complex (D). Experimental data was converted to % inhibition according to Eq. 1 using control values of the assay. Average values of each set of quadruplicates were calculated and utilized for each compound.

Figure 3

Spatial models of TNAP substrates. Space-fill rendering of the molecules of pNPP (A) and CDP-star (B) was generated using ChemBio3D software (CambridgeSoft).

Figure 4

Scaffolds identified in the HTS. TNAP positives were clustered using Accelrys Pipeline Pilot’s Chemistry Component. The following clusters were identified: I – biarylsulfonamide; II – pyrazole; III – triazole; IV - fused pyrazole.

Figure 5

HTS data analysis for biarylsulfonamide series

Figure 6

HTS data analysis for bi-substituted pyrazole series

Figure 7

Representatives of triazole series identified in HTS.

Figure 8

Dose-response curves for representatives of three scaffolds luminescent and colorimetric assays. Compounds MLS-0005718 (A), MLS-0039961 (B), MLS-0067142 (C), MLS-0038949 (D) were tested the HTS luminescent assay containing 100 mM DEA (squares) and colorimetric OD405 (triangles) assays.

Figure 9

Mode of inhibition studies for primary HTS hits in respect to CDP-star substrate. TNAP inhibition with MLS-0005718 (A), MLS-0039961 (B), MLS-0067142 (C), and MLS-0038949 (D) was measured in the presence of 1 uM (squares), 50 uM (triangles) or 300 uM (inversed triangles) CDP-star. The concentration of DEA was equal 100 mM.

Figure 10

Mode of inhibition studies for primary HTS hits in respect to DEA substrate. TNAP inhibition with MLS-0005718 (A), MLS-0039961 (B), MLS-0067142 (C), and MLS-0038949 (D) was measured in the presence of 0 M (squares), 0.1M (triangles) or 1M (inversed triangles) DEA. The concentration of CDP-star was equal 50 uM.

Scheme 1

Catalytic mechanism of alkaline phosphatase reaction (Holtz et al, 1999). Abbreviations: E - alkaline phosphatase enzyme molecule; DO-Pi - substrate molecule; E-Pi – phosphorenzyme (enzyme phosphorylated on Ser-93 in the TNAP sequence of its active site); DOH - product alcohol; E·Pi - non-covalent complex of inorganic phosphate in the active site; AOH – an alcohol molecule, acceptor substrate of transphosphorylation reaction acceptor; AO-Pi – product of transphosphorylation reaction.