1. Identification of novel protease inhibitors active against wild-type and multi-drug-resistant HIV-1 variants including DRV-resistant HIV-1 variants that potentially have a favorable CNS penetration. In the period of this Annual Report, we designed, synthesized, and identified GRL-0739, a novel non-peptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor containing tricycle (cyclohexyl-bis-THF) and a sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (EC50: 0.0019 - 0.0036 mM) with minimal cytotoxicity (CC50: 21.0 mM). GRL-0739 blocked the infectivity and replication of HIV-1NL4-3 variants selected by up to 5 mM concentration of ritonavir or atazanavir (EC50: 0.035 - 0.058 mM). GRL-0739 was also highly active against multi-drug-resistant clinical HIV-1 variants isolated from patients, who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, and HIV-2ROD. The development of resistance against GRL-0739 was substantially delayed compared to APV. The effects of non-specific binding of human serum proteins on GRL-0739's antiviral activity were insignificant. In addition, GRL-0739 showed a favorable blood-brain barrier (BBB) penetration property as assessed using a novel in vitro BBB model. Molecular modeling demonstrated that the tricyclic ring and methoxybenzene of GRL-0739 have a larger surface and seem to make greater van der Waals contact with protease than in the case of darunavir. The present data demonstrate that GRL-0739 has favorable features as a highly active therapeutic with potentially favorable CNS-penetration capability for treating patients infected with wild-type and/or multi-drug-resistant HIV-1 variants and that the newly generated cyclohexyl-bis-THF moiety with methoxybenzene should be critical for the strong binding of GRL-0739 to HIV-1 protease substrate binding site and should have a potential to confer highly favorable anti-HIV-1 potency in designing novel protease inhibitors with greater CNS-penetration profiles. We also identified GRL-09510, a novel non-peptidic HIV-1 protease inhibitor containing a unique polycyclic ring as P2 moiety and a sulfonamide isostere, which is highly active against laboratory HIV-1 strains and primary clinical isolates (EC50: 0.0014 - 0.0028 mM) with minimal cytotoxicity (CC50: 39.0 mM). GRL-09510 blocked the infectivity and replication of HIV-1NL4-3 variants selected by up to 5 mM concentrations of atazanavir, lopinavir, or amprenavir (EC50: 0.0037 - 0.0048 mM). GRL-09510 also maintained its strong antiviral activity against multi-drug-resistant clinical HIV-1 variants isolated from patients, who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, and against HIV-2ROD. The development of resistance against GRL-09510 was substantially delayed compared to that of APV, or integrase inhibitor, raltegravir. Crystallographic analysis demonstrated that the polycyclic ring (P2 moiety) of GRL-09510 has strong hydrogen bond interactions with protease active-site amino acids. Moreover, it has a larger surface and makes greater van der Waals contacts with protease than in the case of darunavir. The data on GRL-09510 demonstrate that the compound has desirable features for treating patients infected with wild-type and/or multi-drug-resistant HIV-1 variants. The structure of the newly designed GRL-09510 is unique, suggesting that the polycyclic-P2 moiety confers highly desirable anti-HIV-1 potency in designing novel protease inhibitors. 2. The C3-substituted tetrahydropyrano-tetrahydofuran-derived protease inhibitors (PIs) exert potent inhibition of the replication of HIV-1 variants highly resistant to various PIs including darunavir. In this subproject, we identified three non-peptidic HIV-1 protease inhibitors (PIs), GRL-015, GRL-085, and GRL-097, which contain a tetrahydropyrano-tetrahydrofuran (Tp-THF) with a hydroxyl moiety at the P2site of the compounds. The three compounds showed potent antiviral activity against a wild-type laboratory HIV-1 strain with 50% inhibitory concentrations (IC50s) of 3.0 to 49 nM and minimal cytotoxicity, with 50% cytotxic concentrations (CC50) for GRL-015, -085, and -097 of 80, 100, and 100 mM, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected in vitro with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multi-drug resistant HIV-1 variants (HIVMDR). Most importantly, a highly darunavir (DRV)-resistant HIV-1 variant, HIV-1DRVRP51, notably less susceptible to DRV over 1,000 times compared to wild-type HIV-1, whereas the three compounds remained active to HIV-1DRVRP51 only with 6.8- to 68-fold reduction. Moreover, in vitro development of drug resistant HIV-1 variants against the three compounds were comparable to or delayed compared to that of DRV, especially highly drug resistant viruses against GRL-085 and -097 were not obtained even by using two different kinds of DRV-resistant HIV-1s as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen-bonds with the polar hydrogen atoms associated with the backbone amide nitrogen atoms of active-site amino acids (Asp29 and Asp30) of HIV-1 protease in a similar fashion to that of bis-THF of DRV. Additionally, strong hydrogen bonding between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings warrant that the three compounds be further studied as possible therapeutic agents for treating individuals harboring wild-type and/or HIVMDR.
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