Discovery and validation of a series of aryl sulfonamides as selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP)

Abstract

We report the characterization and optimization of drug-like small molecule inhibitors of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme critical for the regulation of extracellular matrix calcification during bone formation and growth. High-throughput screening (HTS) of a small molecule library led to the identification of arylsulfonamides as potent and selective inhibitors of TNAP. Critical structural requirements for activity were determined, and the compounds were subsequently profiled for in vitro activity and bioavailability parameters including metabolic stability and permeability. The plasma levels following subcutaneous administration of a member of the lead series in rat was determined, demonstrating the potential of these TNAP inhibitors as systemically active therapeutic agents to target various diseases involving soft tissue calcification. A representative member of the series was also characterized in mechanistic and kinetic studies.

Figure 1

Structures of reported TNAP inhibitors.

Figure 2

Examples of inactive arylsulfonamides which gave insight into required structural elements. TNAP IC₅₀ > 10 μM for all examples.

Figure 3

Elucidation of key requirements of lead TNAP inhibitor 1. TNAP IC₅₀ > 10 μM for compounds 12-17.

Figure 4

The catalytic mechanism of the alkaline phosphatase reaction. See text for details.

Figure 5

Time-dependent mechanistic studies of TNAP inhibition with 1. The enzyme was added with compound 1 serially diluted immediately prior to activity measurement (circle, EI (0:0)) or pre-incubated with the compound for 1h at 12.5-fold the concentrations and then diluted to final concentrations with (diamond, EI (1:1)) or without (inverted triangle, EI (1:0)) an additional 1h incubation after the dilution and prior to activity measurement. The dose-response curves were analyzed using Hill equation; best-fit parameters (IC₅₀ and Hill coefficient n_H) and their standard errors of fit are shown in the Table.

Figure 6

Lineweaver-Burk plots for compound 1 MOA experiments. Inhibition of TNAP was measured in the presence of the following concentrations of 1: 0 μM (open circle), 0.195 μM (closed circle), 0.39 μM (open square), 0.78 μM (closed square), 1.56 μM (open diamond). The concentrations of DEA and CDP-star were equal to 100 mM (A) and 50 μM (B), respectively. The K_i values for compound 1 are 0.34 μM (Fig. 6A) and 0.59 μM (Fig. 6B).