Identification of a selective inhibitor of murine intestinal alkaline phosphatase (ML260) by concurrent ultra-high throughput screening against human and mouse isozymes

Abstract

Alkaline phosphatase (AP) isozymes are present in a wide range of species from bacteria to man and are capable of dephosphorylation and transphosphorylation of a wide spectrum of substrates in vitro. In humans, four AP isozymes have been identified-one tissue-nonspecific (TNAP) and three tissue-specific-named according to the tissue of their predominant expression: intestinal (IAP), placental (PLAP) and germ cell (GCAP) APs. Modulation of activity of the different AP isozymes may have therapeutic implications in distinct diseases and cellular processes. For instance, changes in the level of IAP activity can affect gut mucosa tolerance to microbial invasion due to the ability of IAP to detoxify bacterial endotoxins, alter the absorption of fatty acids and affect ectopurinergic regulation of duodenal bicarbonate secretion. To identify isozyme selective modulators of the human and mouse IAPs, we developed a series of murine duodenal IAP (Akp3-encoded dIAP isozyme), human IAP (hIAP), PLAP, and TNAP assays. High throughput screening and subsequent SAR efforts generated a potent inhibitor of dIAP, ML260, with specificity for the Akp3-, compared to the Akp5- and Akp6-encoded mouse isozymes.

Keywords: Alkaline phosphatase; Enzymes; Inflammatory bowel disease; Inhibitors; Intestinal alkaline phosphatase.

Figure 1

Screening hit

Figure 2

Overall SAR strategy

Figure 3

General structure for dihydrobenzo[d]oxazoles

Figure 4

Potency and selectivity of ML260. The potency of the inhibitor against dIAP (o), PLAP (+) and TNAP (x) was assessed by 16pt dose-response testing in 1536-well format in duplicate in 1xAssay Buffer, containing 100 mM DEA, pH 9.8, 0.02 mM ZnCl₂, 1 mM MgCl₂, and 1:250, 1:1000 and 1:2000 diluted enzymes, respectively, in a total volume of 4 μL/well. The reactions were started by addition of the CDP-star substrate to the final concentrations of 200 μM for the dIAP reaction, and 250 μM for the PLAP and TNAP reactions, with the luminescence intensity measured after 30 min incubation at room temperature.

Figure 5

Selectivity of intestinal isozyme inhibition by ML260. Dose-dependency of inhibition of dIAP (IC₅₀ = 3.8 μM), versus inhibition of EAP (IC₅₀ = 21.1 μM), gIAP (IC₅₀ = 21 μM) and hIAP (IC₅₀ = 27.6 μM), as indicated.

Figure 6

Mechanism of dIAP inhibition by ML260. Double reciprocal plots of residual dIAP activity versus the concentration of pNPP, constructed for each of the indicated ML260 concentrations.

Figure 7

CHO cells expressing dIAP protein, stained with ELF97 in the absence (A) or presence of 10 μM ML260 (C). (B) and (D) are phase contrast views of (A) and (C) respectively. Bar = 50 μm.

Scheme 1

Synthesis of 5, conditions: a. dichloromethane, triethylamine, (70 - 88% yield); b. trifluoroacetic acid, dichloromethane, 0°C warm to RT (100% yield); c. EDC, HOBT, NMM, DMF, (40-55%)