Non-natural nucleotides as modulators of ATPases

ATP is the chemical medium of energy transfer for all living organisms. A common assumption is that the active site of all ATP-binding proteins share similar architecture. This study challenges this assumption by using non-natural nucleotides as chemical probes for exploring the diverse architecture of the ATP binding site. Two systems were used to explore this principle first was the sliding clamp loader ATPase which is responsible for processive DNA replication and second was the ATP binding cassette transporter Pglycoprotein (P-gp) that is involved in multidrug resistance (MDR) phenotype. A library of non-natural nucleotides was used to probe both the T4 bacteriophage and E.coli clamp loaders. One compound, 5-nitroindolyl 2'-deoxyribose triphosphate, was found to selectively inhibit the T4 bacteriophage clamp loader (gp44/62). Mutagenesis studies strongly suggest that specific electrostatic interactions between the nitro moiety and Arg175 of gp44/62 are responsible for the selectivity. Functionally, this inhibition was shown to inhibit processive DNA replication and effectively reduce T4 plaque formation over a lawn of E.coli. Several non-natural nucleosides were submitted to a screening service which identified 5-cyclohexly 2'-deoxyriboside (d5-CHI) as a functional inhibitor of P-gp. From this screen we hypothesized that d5-CHI acts as an ATP competitive inhibitor, however, this compound was shown be a non-transportable substrate of P-glycoprotein that is able to stimulate its ATPase activity and influence the drug stimulated ATPase catalytic efficiency (Vmax/Km) of other important chemotherapeutic drugs. The effects of d5-CHI on drug stimulated P-gp ATPase catalytic efficiency was shown to be in direct correlation with changes in P-gp mediated drug resistance in a P-gp overexpressing MDR cell line. Although the results of this study were contrary to the original hypothesis, d5-CHI proved to be a useful tool for developing a diagnostic method for determining P-gp mediated drug interactions using ATPase activity. This study also demonstrated a unique selectivity of P-gp for its substrates as structurally similar compounds such as 5-cyclohexeneindolyl 2'-deoxyriboside and 5-phenylindolyl 2'-deoxyriboside failed to illicit any interactions with P-gp. Overall, these studies demonstrate the diverse architecture of ATPases and the use of non-natural nucleotides as chemical probes for understanding protein function.