Human immunodeficiency virus type 1 (HIV-1) uses its own reverse transcriptase (RT) to convert its single-stranded RNA genome into a double-stranded DNA copy. Nucleoside RT inhibitors, including zidovudine, didanosine, lamivudine (3TC), and stavudine (d4T), constitute the most important class of antiviral compounds for the treatment of HIV-1 infection (9
). However, the application of these compounds is clinically limited due to their cytotoxicity through inhibition of the host DNA polymerases (4
) and the rapid emergence of drug-resistant viral mutants. Therefore, developing new compounds with reduced cytotoxicity and improved antiviral potency, especially against drug-resistant viral strains, becomes an urgent therapeutic objective.
Our laboratory recently discovered a novel derivative of d4T, namely, 2′,3′-didehydro-3′-deoxy-4′-ethynylthymidine (4′-Ed4T) (Fig. ) (6
). Compared with its parental compound d4T, 4′-Ed4T is fivefold more potent against HIV-1 replication (6
). It also showed much less cytotoxicity than d4T in cell culture studies (6
) because 4′-Ed4TTP had no or only a weak inhibitory effect on major host DNA polymerases (41
). Moreover, 4′-Ed4T was found to be active against many drug-resistant HIV-1 strains (27
). Drug susceptibility studies showed that HIV-1 strains with the M184V single mutation and the P119S/T165A/M184V triple mutations in RT conferred three- to fivefold and 130-fold resistance to 4′-Ed4T, respectively (27
Chemical structures of dT, d4T, and 4′-Ed4T.
Like other nucleoside RT inhibitors, 4′-Ed4T can be phosphorylated in vivo stepwise into its mono-, di-, and triphosphate metabolites by host cell kinases (11
). We showed in steady-state enzymatic analyses that 4′-Ed4TTP, the triphosphate metabolite of 4′-Ed4T, was a substrate of HIV-1 RT (41
). 4′-Ed4TTP inhibited the DNA polymerase activity of RT more efficiently than d4TTP did. We also showed that the inhibition was more effective on DNA replication with RNA template than on that with DNA template. Furthermore, 4′-Ed4TTP was found to inhibit the M184V mutant with threefold-less efficiency than wild-type (wt) RT, consistent with the drug susceptibility studies (27
Steady-state kinetic analysis showed that 4′-Ed4TTP had a sevenfold-lower Ki
value than that of d4TTP, implying the stronger binding of 4′-Ed4TTP to RT. However, steady-state kinetic analysis provides only mechanistic insight into enzyme inhibition that is related to the rate-limiting step. In the case of RT, the slowest step being examined under steady-state conditions is the dissociation of the elongated DNA product from the enzyme (17
). Therefore, this approach is not informative about the detailed interactions of the compound with the RT active site. On the other hand, the pre-steady-state kinetic analysis allows direct examination of the individual steps in the kinetic pathway including binding events, polymerase conformational changes, and the chemical step (14
In the present study, in order to understand the structure-activity relationship for 4′-Ed4TTP, especially the role of its 4′-ethynyl moiety, the pre-steady-state kinetic parameters for 4′-Ed4TMP incorporation by wt RT during DNA- and RNA-dependent DNA polymerization were determined and compared with those of dTMP and d4TMP incorporation. The 3TC-resistant RT mutant M184V was also included in our pre-steady-state kinetic analysis because (i) the structure of RT-primer/template (P/T)-dTTP ternary complex indicated that Met184 constituted part of the nascent base pairing pocket and could affect incoming nucleotide binding (12
); (ii) the M184V viral strain conferred three- to fivefold resistance to 4′-Ed4T (27
); and (iii) more importantly, M184V was the first mutation that emerged in the experiment for selection of resistant virus and perhaps is critical for the development of an additional resistance mutation(s) (27
). Based on these kinetic results and the existing crystal structures, an inhibition mechanism of 4′-Ed4TTP toward RT is proposed.