1. Identification of GRL-04810 and GRL-05010;difluoride-containing nonpeptidic HIV-1 protease inhibitors (PIs) that potently inhibit the replication of multi-PI-resistant HIV-1 in vitro and potentially well penetrate across the blood-brain barrier. One of the sanctuary sites for HIV-1 infection is the central nervous system (CNS). The fact that HIV-1 enters and infects target cells in the CNS represents a significant challenge for the long-term suppression of the virus replication and has been linked to the development of several neurological complications. Although cART has significantly reduced the incidence of HIV-1-associated dementia, the prevalence of CNS disorders such as HIV-1-associated neurocognitive disorders or HAND appears to be increasing as a result of prolonged patient survival and poor antiretroviral drug penetration into the CNS. Furthermore, subtherapeutic drug concentrations in the CNS may facilitate the development of viral resistance. In addition, HIV-1 infection of the CNS may also result in the establishment of a unique viral reservoir, which certain antiretroviral drugs do not have reasonable access to. Moreover, there is evidence that cART is less effective in lowering virus replication in the CNS than in the blood and unfortunately HIV protease inhibitors and several of the nucleoside analogs penetrate only poorly into the CNS, allowing early CNS infection to evolve independently over time in the inaccessible brain reservoir. In the study period covered by this Annual Report, we designed, synthesized, and identified two novel non-peptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-04810 and GRL-05010, containing the structure-based designed privileged non-peptide P2 ligand, bis-tetrahydrofuranylurethane (bis-THF) and a difluoride moiety, both of which are active against a laboratory strain HIV-1LAI (EC50: 0.0008 and 0.003 microM) with minimal cytotoxicity (CC50: 17.5 and 37.0 microM in CD4+ MT-2 cells). The two compounds were active against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after receiving various anti-HIV agents. GRL-04810 and GRL-05010 also blocked the infectivity and replication of each of the HIV-1NL4-3 variants selected by up to 5 microM lopinavir (EC50: 0.03 and 0.03 microM, respectively) and atazanavir (EC50: 0.02 and 0.04 microM, respectively). Moreover, they were active against darunavir-resistant variants (with EC50 values in the range of 0.03-0.034 microM for GRL-04810, and 0.026-0.043 microM for GRL-05010), while darunavir displayed EC50 values between 0.02-0.174 microM. GRL-04810 had a favorable lipophilicity profile as determined with the partition (logP) and distribution coefficients (logD) of -0.14 and -0.29, respectively. The in vitro blood brain barrier (BBB) permeability assay revealed that GRL-04810 and GRL-05010 may have a greater advantage in terms of crossing the BBB than the currently available PIs with apparent penetration indexes of 47.8 x 10-6 and 61.8 x 10-6 cm/s, respectively. The present data demonstrate that GRL-04810 and GRL-05010 exert potent activity against a wide spectrum of HIV-1 variants in vitro and suggest that two fluorine atoms added to their bis-THF moiety may well enhance their penetration across the BBB. 2. Identification of potent PIs containing flexible macrocycles involving P1'-P2'-ligands and P1-P2 ligands. In another approach for the development of protease inhibitors with braod spectrum activity against HIV-1, we have been exploring the design of various macrocyclic HIV-1 protease inhibitors. Recently, we have reported the design of a series of potent PIs that incorporate flexible macrocycles involving P1'-P2'-ligands and P1-P2 ligands to effectively fill in the S1'-S2'and S1-S2 subsites of HIV-1 protease, respectively. The conception of this macrocyclic design evolved from the observation that certain mutations lead to decreased van der Waals interactions and increased the size of the subsite hydrophobic pocket. On the basis of this insight of enzyme flexibility in accommodating alternate packing, we designed flexible macrocycles between the P1'-side chain and a suitable P2'-ligand to fill in the S2'and S1'-subsites. This effort led to a series of potent macrocyclic inhibitors containing the P1-P2-ligands of darunavir. Such compounds incorporated 16- to 19-membered macrocyclic rings between a nelfinavir-like P2 ligand and a tyrosine side chain containing a hydroxyethylamine sulfonamide isostere. All cyclic inhibitors proved to be more potent than their corresponding acyclic counterparts. Saturated derivatives showed slight reduction of potency compared to respective unsaturated derivatives. A compound containing a 16-membered ring as the P1-P2 ligand showed the most potent enzyme inhibitory and antiviral activity. 3. Delayed emergence of HIV-1 variants resistant to 4'-ethynyl- 2-fluoro-2'-deoxy- adenosine: Comparative sequential passage study. Since the development of zidovudine (AZT), the first antiretroviral agent against acquired immune deficiency syndrome (AIDS), a number of therapeutics have been added to the armamentarium in the fight against human immunodeficiency virus type 1 (HIV-1) infection. Currently, one of the most commonly used NRTIs is tenofovir disoproxil fumarate (TDF), which is a prodrug form of tenofovir. Tenofovir has mostly been used as a once-a-day fixed dose tablet combined with emtricitabine (FTC) for treating HIV-1-infected individuals including those who carry HIV-1 strains that are resistant to other existing NRTIs but has also been used for treating patients with hepatitis B virus infection. TDF showed no significant side effects in the initial clinical trials;however, severe acute renal failure was seen in certain patients receiving tenofovir. Moreover, there is a substantial concern that TDF causes nephrotoxicity and osteoporosis with the long-term administration, particularly in aged individuals with HIV-1 infection. Thus, the discovery of the next generation of RT inhibitors, which have less toxicity and do not possess cross-resistance with tenofovir is needed as a component of salvage therapy for patients, who do not tolerate tenofovir or have acquired tenofovir-resistant HIV strains. In this subproject, comparative selection passages against 4'-ethynyl-2'-deoxynucleoside analogs (EdNs), which maintain the 3'-OH in their sugar moiety, lamivudine (3TC), tenofovir (TDF), emtricitabine (FTC), or BMS-986001 (Ed4T) were conducted using a mixture of eleven highly multi-drug-resistant clinical HIV-1 isolates (HIV11MIX) as a starting virus population. Before selection, HIV11MIX was sensitive to EFdA with IC50 of 0.032 microM, less susceptible to TDF and Ed4T with IC50s of 0.57 and 2.6 microM, respectively, and highly resistant to 3TC and FTC with IC50s 10 microM. IC50s of TDF against HIV11MIX exposed to EFdA and TDF for 17 and 14 passages (HIV11MIXEFdA-P17 and HIV11MIXTDF-P14) were 8 and 10 microM, respectively, while EFdA remained active against HIV11MIXEFdA-P17 and HIV11MIXTDF-P14 with IC50s of 0.15 and 0.1 vM, respectively. Both selected variants were highly resistant against zidovudine, 3TC, Ed4T, and FTC (IC50 values 10 microM). The present data demonstrate that HIV11MIX developed resistance more rapidly against 3TC, FTC, TDF, and Ed4T than against EFdA and that EFdA remained substantially active against TDF- and EFdA-selected variants. Thus, EFdA has a favorable resistance profile and represents a potentially promising new generation nucleoside reverse transcriptase inhibitor.
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