Phosphonate inhibitors

Fig. 7a, b. Active site showing Znl-Zn3 and Zn3 - Asp55 distances in complexes of PLCBc with a inorganic phosphate [65] b a phosphonate inhibitor [45]... 

Although the stabilizing interactions between the amino acid side chains of PLC/j, and the choline headgroup are readily apparent in the PLC fc-phosphonate inhibitor complex, it is more difficult to identify contacts between the protein and the acyl chains of the inhibitor [45]. In part this is because thermal motion in the acyl side chains, especially the sn-1 chain, renders them somewhat disordered. Consequently, the measured distances between the side chain carbons... 

One of the questions that is commonly addressed in mechanistic proposals is how is the active site water activated for nucleophilic attack on the phosphodi-ester bond Numerous combinations of amino acid side chains and zinc ions have been proposed for this role, but there has been little consensus. Critical to all the general base hypotheses is a quite reasonable assumption about catalysis by PLC5c The nucleophilic attack on the phosphodiester moiety proceeds via an in-line mechanism resulting in stereochemical inversion of configuration at phosphorus [86]. While this assumption is consistent with the position of the active site water molecules in the PLCBc-phosphonate inhibitor complex [45], it has not yet been established experimentally. This structure provides a detailed picture of how the amino acid side chains of Glul46, Glu4, Asp55, and the zinc ions interact with the phosphonate inhibitor (Fig. 12), so mechanistic hypotheses now have a structural basis. 

Li, Y.-K. Byers, L.D. Phosphonate inhibitors of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. Biochim. Biophys. Acta, 1164, 17-21 (1993)... 

Fig. 8 Mechanism of thermolysin and carboxypeptidase based on X-ray structures of enzymes with bound phosphonate inhibitors.34-37...

Fig. 9 Structure of phospholipase A2 with bound phosphonate inhibitor mimicking the tetrahedral intermediate.38...

Figure 9.3 Comparison of K, values for phosphonate inhibitors of thermolysin with Kulkcat values for the corresponding substrates (Bartlett, 1983 Morgan, 1991). Diagonal lines correspond to slopes of unity.

Bash et al. (1987) applied the thermodynamic perturbation method to complexes of thermolysin with a phosphonamidate [Cbz-Gly -(NH)-Leu-Leu] and the corresponding phosphonate inhibitor [Cbz-Gly -(0)-Leu-Leu]. The perturbation was carried out by using 20 windows, with 2-psec molecular dynamics simulations in each window. Computations were for the ligand in solution and bound to the enzyme. The solvation of the enzyme was represented by a spherical cap of 168 water molecules about the bound inhibitor. The difference in free energy of binding of the two inhibitors was calculated to be 4.38 kcal/mol, to be compared with the experimental value, 4.10 kcal/mol. These calculations point out the importance of solvation effects, which are seen in the 3.4 kcal/mol difference between the NH and O forms of the inhibitor. 

Figure 8 Irreversible inhibitors of proteases. Serine and cysteine proteases can be acylated by aza-peptides, which release an alcohol, but cannot be deacylated due to the relative unreactivity of the (thio) acyl-enzyme intermediate. Reactive carbons, such as the epoxide of E64, can alkylate the thiol of cysteine proteases. Phosphonate inhibitors form covalent bonds with the active site serine of serine proteases. Phosphonates are specific for serine proteases as a result of the rigid and well-defined oxyanion hole of the protease, which can stabilize the resulting negative charge. Mechanism-based inhibitors make two covalent bonds with their target protease. The cephalosporin above inhibits elastase [23]. After an initial acylation event that opens the p-lactam ring, there are a number of isomerization steps that eventually lead to a Michael addition to His57. Therefore, even if the serine is deacylated, the enzyme is completely inactive.

Of course, cell permeability is not always a prerequisite for biologic activity. In one example, Vaghefi et al. (24) proposed that selective phosphonate inhibitors of galactosyltransferase might exhibit activity at the surface of the tumor cell membranes in such cases, a cell permeable inhibitor would be unnecessary. Although no in vivo testing was conducted, phosphonate... 

Crystal Structure of the Open Form of DCL in Complex with a Phosphonate Inhibitor... 

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