Residues Determining the Binding Specificity of Uncompetitive Inhibitors to Tissue-Nonspecific Alkaline Phosphatase

Abstract

Recent data have pointed to TNALP as a therapeutic target for soft-tissue ossification abnormalities. Here, we used mutagenesis, kinetic analysis, and computer modeling to identify the residues important for the binding of known ALP inhibitors to the TNALP active site. These data will enable drug design efforts aimed at developing improved specific TNALP inhibitors for therapeutic use. Introduction:We have shown previously that the genetic ablation of tissue-nonspecific alkaline phosphatase (TNALP) function leads to amelioration of soft-tissue ossification in mouse models of osteoarthritis and ankylosis (i.e., Enpp1 Ϫ/Ϫ and ank/ank mutant mice). We surmise that the pharmacologic inhibition of TNALP activity represents a viable therapeutic approach for these diseases. As a first step toward developing suitable TNALP therapeutics, we have now clarified the residues involved in binding well-known uncompetitive inhibitors to the TNALP active site. Materials and Methods:We compared the modeled 3D structure of TNALP with the 3D structure of human placental alkaline phosphatase (PLALP) and identified the residues that differ between these isozymes within a 12 Å radius of the active site, because these isozymes differ significantly in inhibitor specificity. We then used site-directed mutagenesis to substitute TNALP residues to their respective homolog in PLALP. In addition, we mutagenized most of these residues in TNALP to Ala and the corresponding residues in PLALP to their TNALP homolog. All mutants were characterized for their sensitivity toward the uncompetitive inhibitors L-homoarginine (L-hArg), levamisole, theophylline, and L-phenylalanine. Results and Conclusions:We found that the identity of residue 108 in TNALP largely determines the specificity of inhibition by L-hArg. The conserved Tyr-371 is also necessary for binding of L-hArg. In contrast, the binding of levamisole to TNALP is mostly dependent on His-434 and Tyr-371, but not on residues 108 or 109. The main determinant of sensitivity to theophylline is His-434. Thus, we have clarified the location of the binding sites for all three TNALP inhibitors, and we have also been able to exchange inhibitor specificities between TNALP and PLALP. These data will enable drug design efforts aimed at developing improved, selective, and drug-like TNALP inhibitors for therapeutic use.

Supplemented Chondroitin Sulfate and Hyaluronic Acid Suppress Mineralization of the Chondrogenic Cell Line, ATDC5, <i>via</i> Direct Inhibition of Alkaline Phosphatase

“…Hydrolysis of pyrophosphate to inorganic phosphate by ALP underlies the mineralization of chondrocytes. [34][35][36] Our data suggest that CS and HA regulate the differentiation, i.e., mineralization of chondrocytes via direct suppression of ALP activity. Such control cells with ITS were confirmed to be AB-positive and some cartilage differentiation gene markers (Sox9, Runx2, Col2 and Aggrecan) of the cell were enhanced at this stage (data not shown).…”

Tissue-nonspecific Alkaline Phosphatase Is Required for the Calcification of Collagen in Serum

“…Previous studies have shown that the initial step in serum-induced apatite formation is highly dependent on the concentration of calcium and phosphate in solution, and will not continue to occur when the calcium phosphate ion product falls below about 2.5 mM 3 (1, 2). (26)) and levamisole (an uncompetitive inhibitor (27,28)) completely prevented the uptake of serum calcium by bone, and Table 1 shows that bone samples incubated in serum with either beryllium or levamisole had no detectable calcium or phosphate, and did not stain with Alizarin red.…”

Cellular function and molecular structure of ecto-nucleotidases

“…A unique feature of the active site of the mammalian enzyme in comparison to EcAP is the presence of Y367 at a distance of 6.1 Å to the phosphate ion. This tyrosine, which protrudes from the neighboring subunit into the active site, has been shown to be involved in the uncompetitive inhibition of mammalian APs [532,593]. To characterize the structural basis of the allosteric behavior and uncompetitive inhibition of PLAP, co-crystal structures with various ligands influencing the catalytic activity of the enzyme have been determined [594].…”