1. Identification of novel protease inhibitors active against wild-type and multi-drug-resistant HIV-1 variants including DRV-resistant HIV-1 variants. We have been focusing on the design and synthesis of non-peptidyl protease inhibitors (PIs) that are potent against HIV-1 variants resistant to the currently approved PIs in collaboration with Professor Arun K.Ghosh of Purdue University and identified more than 30 potent protease inhibitors and PDIs. One such anti-HIV-1 agent, darunavir (DRV), containing a structure-based designed privileged nonpeptidic P2 ligand, 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF), has been approved as a first-line therapeutic agent for the treatment of individuals, who are infected with HIV-1. We examined and characterized the non-peptidic HIV-1 protease inhibitors GRL-0519 and its stereoisomer, GRL-0529, both of which contain the tris-THF moiety and a sulfonamide isostere. We demonstrated that GRL-0519containing tris-tetrahydrofuranylurethane (tris-THF) and a sulfonamide isostere, is highly potent against laboratory HIV-1 strains and primary clinical isolates (EC50: 0.0005 - 0.0007 microM) with minimal cytotoxicity (CC50: 44.6 microM). GRL-0519 blocked the infectivity and replication of HIV-1NL4-3 variants selected by up to 5 microM concentration of ritonavir, lopinavir, or atazanavir (EC50: 0.0028 - 0.0033 microM). GRL-0519 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients, who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, highly darunavir (DRV)-resistant variants, and HIV-2ROD. The development of resistance against GRL-0519 was substantially delayed compared to other PIs including APV and DRV. The effects of non-specific binding of human serum proteins on GRL-0519's antiviral activity were insignificant. Our analysis of the crystal structures of GRL-0519 (3OK9) and DRV (2IEN) with protease suggested that the tris-THF moiety, compared to the bis-THF moiety present in DRV, has greater water-mediated polar interactions with key active site residues of protease and that the tris-THF moiety and para-methoxy group effectively fill the S2 and S2'binding pockets, respectively, of the protease. The data demonstrate that GRL-0519 has highly favorable features as a potential therapeutic agent for treating patients infected with wild-type and/or multi-PI-resistant variants and that the tris-THF moiety is critical for strong binding of GRL-0519 to the HIV protease substrate binding site and appears to be responsible for its favorable antiretroviral characteristics. 2. Study of the structure-activity relationship studies using various biological/antiviral assays and crystallography. In this project, GRL007 and GRL008, two structurally related nonpeptidic human imicrounodeficiency virus type-1 (HIV-1) protease inhibitors (PI) containing 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) as the P2 moiety and a sulfonamide isostere consisting of benzene carboxylic acid and benzene carboxamide as the P2'moiety, respectively, were evaluated for their antiviral activity and interactions with wild-type protease (PRWT). Both GRL007 (Ki: 12.7 pM with PRWT) and GRL008 (Ki: 8.9 pM) inhibited PRWT with high potency, in-vitro. X-ray crystallographic analysis of PRWT in complex with GRL007 or GRL008 showed that the bis-THF moiety of both compounds has three direct polar contacts with the backbone amide nitrogen atoms of Asp29 and Asp30 of PRWT. The P2'moiety of both compounds showed one direct contact with the backbone of Asp30'and a bridging polar contact with Gly48'through a water molecule. Cell-based antiviral assays showed that GRL007 was inactive (EC50: 1 MICROM) while GRL008 was highly active (EC50: 0.04 MICROM) against wild-type HIV-1. The HPLC/mass spectrometry-based cellular uptake assay showed 8.1- and 84-fold higher intracellular concentrations of GRL008 compared to that of GRL007 in human MT-2 and MT-4 cell extracts, respectively. Thus, GRL007, in spite of its favorable enzyme inhibitory activity and protease binding profile, exhibited lack of antiviral activity in cell-based assays most likely due to its compromised cellular uptake associated with its P2'benzene carboxylic acid moiety. The features of anti-HIV-1 potency, favorable toxicity, and binding profile of GRL008 suggest that further optimization of the P2'moiety may improve its antiretroviral features. 3. Characterization of the profiles of EFdA for potential clinical development. A number of individuals who received currently available nucleoside reverse transcriptase inhibitors (NRTIs) have developed resistant HIV-1 variants. However, there are only a few NRTIs that are on the pipeline as of today. The development of novel NRTIs, which are more potent and less toxic, and prevent or delay the emergence of resistant HIV-1 variants, is needed. We have previously identified 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), which is highly potent against various wild-type and multi-drug-resistant HIV-1 variants and and possess favorable pharmacokinetic/pharmacodynamic profiles potentially allowing once daily (QD) dosing schedule. In the period covered by this report, comparative sequential passages were conducted using MT4 cells and a mixture of 11 highly multi-drug-resistant clinical isolates of HIV-1 (HIV11MIX) as a starting virus population in the presence of increasing concentrations of EFdA, tenofovir (TDF), emtricitabine (FTC), or festinavir (Ed4T). Before selection, HIV11MIX was sensitive to EFdA with the IC50 value of 0.032 microM, was less susceptible to TDF, and Ed4T with the IC50 values of 0.57 and 2.6 microM, respectively, and was highly resistant to 3TC and FTC with the IC50 values 10 microM. It took 10 passages for the concentration of FTC and Ed4T to reach 10 microM, under which HIV11MIX3TC-P10 and HIV11MIXFTC-P10 robustly replicated. It took 20 passages for TDF to reach 10 microM. HIV11MIXTDF-P10 relatively poorly replicated;however, HIV11MIXTDF-P20 had regained robust replicability. It took more than 20 passages for EFdA to reach a relatively low concentration, 3 microM. Clonal HIV11MIXEFdA-P10 had compromised replicability compared to a recombinant clonal virus HIVNL4-3, while HIV11MIXEFdA-P20 had gained moderate replicability. The IC50 values of TDF against HIV11MIX exposed to EFdA and TDF for 14 passages (HIV11MIXEFdA-P14 and HIV11MIXTDF-P14) were 8 and 10 microM, while EFdA remained active against HIV11MIXEFdA-P14 and HIV11MIXTDF-P14 with the IC50 values of 0.15 and 0.1 microM, respectively. Both HIV11MIXEFdA-P14 and HIV11MIXTDF-P14 were highly resistant against AZT, 3TC, Ed4T, and FTC (IC50 values 10 microM). Out of 11 clinical isolates, HIVC had become predominant (70%) in HIV11MIX before the selection and contained M41L/E44D/D67N/T69D/M184V/L210W/T215Y/K219N. HIV11MIXEFdA-P10 and HIV11MIXTDF-P10 contained M41L/D67del/T69G/K70R/L74I/V75T(A) /M184V/T215F/K219Q. Most of these amino acid changes appeared to have been acquired from a clinical isolate, HIVB, through homologous recombination. The present data strongly suggest that HIV11MIX population develops resistance more rapidly against FTC, TDF and Ed4T than against EFdA, that EFdA resistance development is significantly delayed, and that EFdA remains substantially active against TDF- and EFdA-selected variants. Thus, EFdA has a unique resistance profile and represents a promising new generation RTI.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
Application #
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
National Cancer Institute Division of Clinical Sciences
Zip Code