Abstract
The tissue-nonspecific alkaline phosphatase (TNAP) isozyme is centrally involved in the control of normal skeletal mineralization and pathophysiological abnormalities that lead to disease states such as hypophosphatasia, osteoarthritis, ankylosis and vascular calcification. TNAP acts in concert with the nucleoside triphosphate pyrophosphohydrolase-1 (NPP1) and the Ankylosis protein to regulate the extracellular concentrations of inorganic pyrophosphate (PPi), a potent inhibitor of mineralization. In this review we describe the serial development of two miniaturized high-throughput screens (HTS) for TNAP inhibitors that differ in both signal generation and detection formats, but more critically in the concentrations of a terminal alcohol acceptor used. These assay improvements allowed the rescue of the initially unsuccessful screening campaign against a large small molecule chemical library, but moreover enabled the discovery of several unique classes of molecules with distinct mechanisms of action and selectivity against the related placental (PLAP) and intestinal (IAP) alkaline phosphatase isozymes. This illustrates the underappreciated impact of the underlying fundamental assay configuration on screening success, beyond mere signal generation and detection formats.
Highlights
Alkaline phosphatases (E.C.3.1.3.1) (APs) are dimeric enzymes, present in most organisms [1]where they catalyze the hydrolysis of phosphomonoesters
tissue-nonspecific alkaline phosphatase (TNAP) is centrally involved in mechanisms that control normal skeletal mineralization and pathophysiological abnormalities that lead to disease states such as hypophosphatasia, osteoarthritis, ankylosis and vascular calcification [2]
TNAP acts in concert with the nucleosidetriphosphate pyrophosphohydrolase-1 (NPP1) and the Ankylosis protein to regulate the extracellular concentrations of inorganic pyrophosphate (PPi), a potent inhibitor of hydroxyapatite formation at concentrations normally found in plasma [3,4,5]
Summary
Alkaline phosphatases (E.C.3.1.3.1) (APs) are dimeric enzymes, present in most organisms [1]. In this article we describe the serial development of two miniaturized high-throughput screens (HTS) for TNAP inhibitors that differ in both signal generation and detection formats, but more critically in the concentrations of a terminal alcohol acceptor used These assay improvements allowed the rescue of the initially unsuccessful screening campaign against a large small molecule chemical library, but enabled the discovery of several unique classes of molecules with distinct mechanisms of action and selectivity against the related PLAP and IAP isozymes. This illustrates the underappreciated impact of the underlying fundamental assay configuration on screening success, beyond mere signal generation and detection formats. Activators of TNAP function may be useful for the treatment of milder forms of hypophosphatasia, as the therapeutic use of TNAP activators might sufficiently enhance residual TNAP activity to help resolve or prevent the rickets/osteomalacia characteristic of the disease and may be useful as a means of increasing bone mineral density in patients with osteoporosis
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