Nonspecific inhibitors

Figure 11.9 A diagram of the active site of chymotrypsin with a bound inhibitor, Ac-Pro-Ala-Pro-Tyr-COOH. The diagram illustrates how this inhibitor binds in relation to the catalytic triad, the strbstrate specificity pocket, the oxyanion hole and the nonspecific substrate binding region. The Inhibitor is ted. Hydrogen bonds between Inhibitor and enzyme are striped. (Adapted from M.N.G. James et al., /. Mol. Biol. 144 43-88, 1980.)...

The C-terminal part is green. The catalytic triad Asp 32, His 64, and Ser 221 as well as Asn 15S, which forms part of the oxyanion hole are shown in purple. The main chain of part of a polypeptide Inhibitor is shown in red. Main-chain residues around 101 and 127 (orange circles) form the nonspecific binding regions of peptide substrates. 

Figure 11.14 Schematic diagram of the active site of subtilisin. A region (residues 42-45) of a bound polypeptide inhibitor, eglin, is shown in red. The four essential features of the active site— the catalytic triad, the oxyanion hole, the specificity pocket, and the region for nonspecific binding of substrate—are highlighted in yellow. Important hydrogen bonds between enzyme and inhibitor are striped. This figure should be compared to Figure 11.9, which shows the same features for chymotrypsin. (Adapted from W. Bode et al., EMBO /.

Kinetic inhibitors for hydrate formation may also be effective in preventing scale deposition [1627]. This may be understood in terms of stereospecific and nonspecific mechanisms of scale inhibition. 

Enzyme solutions can be stabilized using sugars, polyhydric alcohols, polymers, or salts [65]. These compounds affecting the enzyme stability are ligands (substate, product, inhibitor, coenzymes) or nonspecific additives. 

From the preceding sections, it is clear that chemokines are important players in atherosclerotic disease and, as such, are being considered as possible targets in the treatment of this prevalent inflammatory condition. Under consideration at this time are both traditional nonspecific therapies [e.g., 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, fibrates, etc.], as well as chemokine specific approaches (142). 

FIGURE 5.4 In this illustration of a competition experiment, a fixed concentration of radioligand, in the absence of inhibitor, produces specific binding of B0. The specific binding in the presence of a competitive inhibitor is denoted by Bt A constant amount of nonspecific binding is assumed to be present. The concentration of inhibitor that reduces specific binding by 50% is referred to as the IC50. 

We have found that many compounds identified in our screen are nonspecific inhibitors of luciferase enzyme activity. To eliminate these, we test the hits in a luciferase enzyme-based counterscreen. Firefly and renilla luciferase are produced in vitro by programming Krebs-2 extracts with FF/HCV/Ren mRNA and allowing the translations to proceed at 30° for 1 h. Ten microliters are then pipetted into a 96-well plate and compound is added to a final concentration of 20 /iM (1% DMSO). Luciferase activity is then determined as described previously in step 3. Since compound is added only after the translation reaction is complete, inhibitors of translation should not score positively in this assay. Typically, a 1-ml in vitro translation reaction is sufficient to screen 45 candidate hits in duplicate for nonspecific luciferase inhibitory activity. Compounds that inhibit in this counterscreen are eliminated from future analysis. 

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