""""""""In this report period, we maintained our long-standing research line, development of anti-HIV drugs, however, we particularly focused our effort to develop antiretroviral agents which resist the emergence of drug-resistant HIV-1 variants, in response to the rapidly growing problem, drug failure in patients with HIV-1 infection. Over the past three years, we designed and synthesized ~300 potential HIV protease inhibitors in collaborations with intra- and extramural investigators and tested them for antiviral activity against HIV-1 in vitro and identified JE-2147, a novel allophenylnorstatine (apns)-containing dipeptide HIV protease inhibitor (PI). JE-2147 is potent against a wide spectrum of HIV-1, HIV-2, SIV, and clinical HIV-1 strains, and in particular, against multi-PI-resistant HIV-1 strains in vitro. Drug-resistant clinical HIV-1 strains, isolated from seven patients who had failed 9-11 different anti-HIV therapeutics after 32-83 months, had a variety of drug-resistance-related amino acid substitutions and were highly and invariably resistant to all the currently available anti-HIV agents. JE-2147 was, however, extremely potent against all such drug-resistant strains. The emergence of JE 2147-resistant HIV-1 variants in vitro was substantially delayed. Structural analysis revealed that the presence of a flexible P2U moiety is important for the potency of JE-2147 toward wild type and mutant viruses. These data suggest that the use of flexible components may open a new avenue for designing PIs which resist the emergence of PI-resistant HIV-1. It is of note that JE-2147 is currently undergoing pre-clinical and clinical development in the US. The accumulating data suggest that the development of HIV-1 variants with reduced susceptibility to nucleoside reverse transcriptase inhibitors (NRTI) is related to clinical deterioration in patients receiving combination therapy with multiple RT inhibitors. We identified a set of novel mutations [Ala-62.Val(A62V), V75I, F77L, F116Y, and Q151M] in the polymerase domain of RT, which confers on the virus a reduced sensitivity to multiple NRTI in six HIV-1 isolates (17%) from 36 patients, most receiving long-term combination therapy at the DCS, NCI. Q151M was among the first of the substitutions to appear. The loss of initial clinical benefits brought about by combination chemotherapy coincides with the appearance of the MDR mutations. Using a set of recombinant infectious clones, we also studied whether the replication kinetics of HIV-1 was altered when the virus acquired a set or subsets of the five mutations in the presence of drugs. We found a resistance pathway in which HIV 1MDR develops through one or more mutations, which, however, sacrifice replicative capability, then HIV-1 finally acquires optimal replication-competence by additional mutations. In response to the emergence of HIV-1MDR, we have shown that HIV-1 does not readily develop resistance toward a novel NRTI, 2'-b-fluoro-2',3'-dideoxyadenosine (FddA), and MDR remains sensitive to this compound. Phase I/II clinical trials of FddA are underway in collaboration with Dr. Robert Yarchoan's group at DCS. We have seen that HIV-1 in those receiving FddA has not developed resistance to F-ddA. We are also developing several anti-HIV agents including novel NRTIs and PIs. Among them are cyclopropane-based and phenylphosphoralaninated NRTIs, designated QYL-685 and QYL-609. Several PIs which, like JE-2147, are potent against a panel of highly PI-resistant clinical HIV-1 variants and do not allow HIV-1 to develop resistance, are being developed.""""""""
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