Exploiting the Bridged Binding Site

The feasibility of designing inhibitors for resistance enzymes that target the binding sites of both the cofactor and the aminoglycoside is based on the enzyme mechanism of APH(30)s. Whereas APH(30)-Ia functions by a random equilibrium BiBi mechanism (Siregar et al. 1995), APH(30)-IIIa catalyses its reaction by the Theorell-Chance mechanism, a form of ordered BiBi mechanism (McKay and Wright 1995). Both mechanisms require that all substrates must be present in the active site prior to catalysis. Using this approach, tethered derivatives of adenosine and the aminoglycoside neamine have been synthesized (Liu et al. 2000). These bisubstrate analogues are made by covalently linking the 50-hydroxyl of adenosine to the 30-hydroxyl of neamine via methylene linkers of various lengths (Fig. 12). Appropriate linker lengths that very nearly span the distance between the ATP and the aminoglycoside binding sites should manifest themselves by showing strong inhibition of the resistance enzyme. When tested against the APH(30)-Ia and APH(30)-IIIa, compounds with linkers of 6-7 carbons in length were found to be the most potent competitive inhibitors of both ATP and kanamycin A.

Fig. 12 A tethered derivative of adenosine and neamine that targets both the nucleotide-and substrate-binding sites of aminoglycoside-modifying enzymes

These bisubstrate inhibitors must contain many elements required for binding both regions of the active site of aminoglycoside kinases. The specificity of the molecule is increased and the problem of cross-reactivity with host protein kinases can be circumvented. Nevertheless, the breadth of activity of these molecules is reduced, since they are able to bind only those resistance factors that utilize ATP. In addition, the requirement for sufficient specificity in both binding pockets means that compounds that are developed to meet these conditions are likely to be large. Such compounds are unlikely to be effective therapeutic agents, due to issues associated with membrane transport of large molecules (Burk and Berghuis 2002).

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