Substrate-competitive Inhibitors

The substrate-binding site seems to have obvious advantages over the ATP-binding site as a target for inhibiting kinase activity. First, substrate-binding inhibitors are not affected by the high ATP concentration found in cells. Second, the substrate- 

Substrate-competitive inhibition is a well known strategy for targeting enzymes, which has been applied successfully in enzyme classes such as the proteases. Nevertheless, its use for kinase inhibition has met with little success. One of the reasons is the rather stretched substrate pocket of kinases. Kinases are likely to use additional binding pockets, which are not located in the immediate environment of the active site [16, 17]. Therefore, kinases lack the specific hydrophobic pockets that could serve as targets for peptidomimetics, as occurs with HIV protease or thrombin. 

An obvious drawback of this approach is that it involves large peptide-like molecules, which are notorious for problems with oral absorption, stability, and cell penetration. 

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Competitive inhibitors bind only to the free enzyme and to the same site as the substrate. Competitive inhibitors are molecules that usually look like the substrate but can t undergo the reaction. At an infinite concentration of the substrate (1/[S] = 0), the competitive inhibitor cannot bind to the enzyme since the substrate concentration is high enough that there is virtually no free enzyme present. 

ALTERNATIVE PRODUCT INHIBITION ABORTIVE COMPLEXES ALTERNATIVE SUBSTRATES COMPETITIVE INHIBITOR ABORTIVE COMPLEXES MAPPING SUBSTRATE INTERACTIONS USING KINETIC DATA MEMBRANE TRANSPORT ENERGY OF ACTIVATION Old... 

AG 1024 has been extensively studied as an IGFR inhibitor [70] and is a substrate competitive inhibitor of this kinase [71]. AG1024 also inhibits other kinases including c-Kit [72]. Additional studies will be needed, including a direct measurement of Abl activity and possible subsequent testing against the imatinib resistant Abl point mutations, to ascertain the possible therapeutic utility of AG 1024. 

General aspects of enzymatic reactions cateuLyzed by kinases are briefly mentioned. Many alternate substrates, competitive inhibitors and affinity labels based either on the structure of ATP or on the structure of the non-ATP kinase substrates are described. Several examples are presented that should be of particular interest to the medicinal chemist. Finally, the design of an affinity label for creatine kinase is reviewed as an example of how such information can be used in the search for agents directed at an enzyme s active site. 

Substrate-competitive inhibitors. The binding of chelating agents like 2,2 -bipyridine and... 

Imidazole also acts as a substrate-competitive inhibitor, forming both binary complexes with LADH, and ternary complexes in the presence of coenzyme. X-Ray studies show that imidazole also binds to the. catalytic zinc by displacing the water molecule.1361 The presence of imidazole at the active site also enhances the rate of carboxymethylation14658 of Cys-46 with both iodoacetate and iodoacetamide.1420 This enhancement of alkylation has become known as the promotion effect .1421 Imidazole promotion also improves the specificity of the alkylation.1422 Since Cys-46 is thought to be alkylated as a metal-thiol complex, imidazole, on binding the active site metal, could enhance the reactivity by donating a electrons to the metal atom, which distributes the increased electron density further to the other ligands in the coordination sphere. The increased nucleophilicity of the sulfur results in promoted alkylation.1409... 

Another approach toward effective substrate-competitive inhibitors was recently reported by Hubbard et al. [20]. Linking the known IRS-727 octadecapeptide substrate to a stable ATP mimic resulted in a combined ATP- and substrate-competitive inhibitor (compound 1, Figure 7.7) having a K of 370 nM for IRK. 

It has been known since the early studies of Kearney (192) that succinate dehydrogenase undergoes reversible activation by substrates, competitive inhibitors, and phosphate. The activation of succinate dehydrogenase was shown to be a characteristic of both the soluble and particle-bound enzyme and a slow process requiring many minutes of incubation with the activator at ambient or higher temperatures (activation energy = 31-33 kcal/mole). It has been suggested that the enzyme exists in a free equilibrium between the unactivated and the activated forms, and that the activator interacts with the latter and establishes a new equilibrium in favor of the activated state of the enzyme (23, 25, 193 see also 194 for an expanded mechanism). 

Enzyme ligands more often lead to the inhibition of the enzyme activity, binding the active site with competition with the substrate (competitive inhibitors) or to allosteric sites (non-competitive inhibitors). Activation of an enzyme is more difficult to proceed unless giving or generating an excess of substrate or co-substrate. However some drugs are known to activate enzymes by direct binding, that is, forskolin for adenylyl cyclase. 

However, the findings (171) that binary complexes of zinc-free enzyme and coenzyme bind substrates and substrate competitive inhibitors such as isobutyramide cannot be taken as evidence that zinc does not participate in the catalytic action. Several SH groups are probably oxidized in the zinc-free enzyme (170). Evidence has also been presented (172) of other structural differences compared to the catalytically active enzyme. Thus, artificial binding to this enzyme with no relevance to the catalytic action is not unlikely. 

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