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These investigators described the preparation and characterization of several analogs (8) of kemptide. Although the IC50 values of 8a, 8b, and 8c (935 |iM, 226 |iM, and 68 |iM, respectively) are modest, these inhibitors are significantly more effective than the simple Ala-containing analog Leu-Arg-Arg-Ala-Ala-Leu-Glu. The authors found that 8 displays a competitive inhibition pattern versus variable ATP, but such a pattern was not observed with respect to variable peptide substrate. One might expect that a bisubstrate analog would exhibit competitive patterns versus both ATP and phosphory-latable peptide. However, the absence of double competitive behavior does not necessarily rule out the two-site binding model. Strictly speaking, competitive behavior is observed for an inhibitor only if that inhibitor and the corresponding substrate bind in a mutually exclusive fashion to the same enzyme form. PKA is known to exhibit a primarily ordered mechanism with ATP binding first (Whitehouse et al. 1983). Consequently, one would expect compound 8 and ATP to associate with the same enzyme form, namely the free enzyme, and thereby exhibit competitive behavior. By contrast, given the nature of the ordered mechanism, peptide substrate preferentially coordinates to the enzyme-ATP complex, which would thereby rule out a competitive pattern with 8.

Recently Uri and his colleagues have described a series of bisubstrate analogs that dispenses with the phosphoric anhydride portion of the ATP moiety (Loog et al. 1999). These investigators employed an adenosine-50-carbox-ylic acid derivative as the ATP mimic which, using a variety of linkers, was appended to the N-terminus of an arginine rich peptide (9). The most effective analogs displayed IC50 values of between 100 and 300 nM for PKA and PKC. These bisubstrate analogs have been used to affinity-purify protein kinases (Loog et al. 2000). In addition, membrane-permeable fluorophore-labeled bisubstrate derivatives have been prepared (Uri et al. 2002; Viht et al. 2003).

In a departure from the ATP-based bisubstrate strategy, Sergheraert and colleagues designed (ATP mimics)-linker-substrate analogs (Ricouart et al. 1991). Isoquinoline and naphthalene sulfonic acid derivatives served as ATP replacements. The most potent of the several derivatives prepared was compound 10.

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