A Novel Tricyclic Ligand-Containing Nonpeptidic HIV-1 Protease Inhibitor, GRL-0739, Effectively Inhibits the Replication of Multidrug-Resistant HIV-1 Variants and Has a Desirable Central Nervous System Penetration Property In Vitro

Abstract

We report here that GRL-0739, a novel nonpeptidic HIV-1 protease inhibitor containing a tricycle (cyclohexyl-bis-tetrahydrofuranylurethane [THF]) and a sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC₅₀], 0.0019 to 0.0036 μM), with minimal cytotoxicity (50% cytotoxic concentration [CC₅₀], 21.0 μM). GRL-0739 blocked the infectivity and replication of HIV-1_NL4-3 variants selected by concentrations of up to 5 μM ritonavir or atazanavir (EC₅₀, 0.035 to 0.058 μM). GRL-0739 was also highly active against multidrug-resistant clinical HIV-1 variants isolated from patients who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, as well as against the HIV-2_ROD variant. The development of resistance against GRL-0739 was substantially delayed compared to that of amprenavir (APV). The effects of the nonspecific binding of human serum proteins on the anti-HIV-1 activity of GRL-0739 were insignificant. In addition, GRL-0739 showed a desirable central nervous system (CNS) penetration property, as assessed using a novel in vitro blood-brain barrier model. Molecular modeling demonstrated that the tricyclic ring and methoxybenzene of GRL-0739 have a larger surface and make greater van der Waals contacts with protease than in the case of darunavir. The present data demonstrate that GRL-0739 has desirable features as a compound with good CNS-penetrating capability for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants and that the newly generated cyclohexyl-bis-THF moiety with methoxybenzene confers highly desirable anti-HIV-1 potency in the design of novel protease inhibitors with greater CNS penetration profiles.

INTRODUCTION

Combination antiretroviral therapy (cART) has had a major impact on the AIDS epidemic in industrially advanced nations. Recent analyses have revealed that that mortality rates for HIV-1-infected persons have come close to those of the general population (1–4). Moreover, it is noteworthy that an increase in treatment from previous years, as more people are receiving cART, has brought about a >30% decline in the number of new infections, particularly in developing areas, including sub-Saharan countries (5). However, no eradication of human immunodeficiency virus type 1 (HIV-1) currently appears to be possible, in part due to the viral reservoirs remaining in the blood and infected tissues. Moreover, we have encountered a number of challenges in bringing about the optimal benefits of the currently available therapeutics for AIDS and HIV-1 infection to individuals receiving cART (6–8). These include (i) drug-related toxicities, (ii) an inability to fully restore normal immunologic functions once individuals have developed full-blown AIDS, (iii) the development of various cancers as a consequence of survival prolongation, (iv) the flaring up of inflammation in individuals receiving cART or immune reconstruction syndrome (IRS), and (v) an increased cost of antiviral therapy. Such limitations and flaws of cART are exacerbated by the development of drug-resistant HIV-1 variants (8–13), although the recent first-line cART with boosted-protease inhibitor-based regimens has made the development of HIV-1 resistance relatively less likely over an extended period of time (14).

In theory, successful antiviral drugs exert their virus-specific effects by interacting with viral receptors, virally encoded enzymes, viral structural components, viral genes, or their transcripts, without disturbing the cellular metabolism or function. However, at present, no antiretroviral drugs or agents are likely to be completely specific for HIV-1 or to be devoid of toxicity or side effects in the treatment of AIDS. This is a critical issue, because patients with AIDS and its related diseases will have to receive antiretroviral therapy for a long period of time, perhaps for the rest of their lives. Thus, the identification of new classes of antiretroviral drugs that have a unique mechanism(s) of action and produce no or minimal side effects remains an important therapeutic objective.

We have been focusing on the design and synthesis of nonpeptidyl protease inhibitors that are active against HIV-1 variants resistant to the currently approved HIV-1 protease inhibitors. One such anti-HIV-1 agent, darunavir (DRV), which contains a structure-based designed privileged nonpeptidic P2 ligand, 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) (15–17), has been approved as a first-line therapeutic agent for the treatment of individuals who are infected with HIV-1. We also recently reported that a few HIV protease inhibitors, GRL-0519 containing tris-THF (18, 19) and GRL-04810 and GRL-05010, show good CNS penetration in a blood-brain barrier (BBB) reconstruct model in vitro (20). In the present work, we examined and characterized the nonpeptidic HIV-1 protease inhibitor GRL-0739 (21), which contains the cyclohexyl-bis-THF moiety and a sulfonamide isostere (Fig. 1). We found that GRL-0739 exerts strong activity against a wide spectrum of laboratory HIV-1 strains and primary clinical isolates, including multi-HIV-1 protease inhibitor-resistant variants, with minimal cytotoxicity. In addition, GRL-0739 was active against HIV-2_ROD and the HIV-1 isolates we examined. We also selected drug-resistant HIV-1 variants with GRL-0739 by propagating a laboratory wild-type HIV-1_NL4-3 in MT-4 cells in the presence of increasing concentrations of GRL-0739, and we determined the amino acid substitutions that emerged under the pressure of GRL-0739 in the protease-encoding region. In addition, we evaluated the nonspecific binding effects of physiologic human serum proteins on the anti-HIV-1 activity of GRL-0739. Finally, GRL-0739 showed a desirable BBB penetration property in a novel in vitro BBB model.

FIG 1.

Structures of GRL-0739, amprenavir, and darunavir. M.W., molecular weight.

MATERIALS AND METHODS

Cells and viruses.

MT-2 and MT-4 cells were grown in RPMI 1640-based culture medium supplemented with 10% fetal calf serum (FCS) (JRH Biosciences, Lenexa, MD), 50 U/ml penicillin, and 100 μg/ml kanamycin. These cells were obtained from the National Cancer Institute (NCI)/NIH. The following HIV-1 viruses were employed for the drug susceptibility assay (see below): HIV-1_LAI, HIV-1_NL4-3, HIV-2_ROD, and HIV-1_ERS104pre, a clinical HIV-1 strain isolated from a drug-naive patient with AIDS (22), as well as six HIV-1 clinical strains that were originally isolated from patients with AIDS who had received 9 to 11 anti-HIV-1 drugs over the past 32 to 83 months and were genotypically and phenotypically characterized as multi-protease inhibitor-resistant HIV-1 variants (23, 24). All primary HIV-1 strains were passaged once or twice in 3-day-old phytohemagglutinin-activated peripheral blood mononuclear cells (PHA-PBMCs), and the virus-containing culture supernatants were stored at −80°C until use as sources of infectious virions.

Antiviral agents and human serum proteins.

Roche Products Ltd. (Welwyn Garden City, United Kingdom) and Abbott Laboratories (Abbott Park, IL) kindly provided saquinavir (SQV) and ritonavir (RTV), respectively. Amprenavir (APV) was received as a courteous gift from GlaxoSmithKline, Research Triangle Park, NC. Lopinavir (LPV) was kindly provided by Japan Energy, Inc., Tokyo, Japan. Atazanavir (ATV) was a contribution from Bristol Myers Squibb (New York, NY). Darunavir (DRV) was synthesized as previously described (25). Human serum albumin (HSA) and α₁-acid glycoprotein (AAG) were purchased from Sigma-Aldrich (St. Louis, MO). Raltegravir (RAL), emtricitabine (FTC), and maraviroc (MVC) were kindly provided by Kenji Maeda (NCI/NIH).

Drug susceptibility assay.

The susceptibilities of HIV-1_LAI or HIV-2_ROD to various drugs were determined as previously described (18). Briefly, MT-2 cells (10⁴/ml) were exposed to 100 50% tissue culture infectious doses (TCID₅₀) of HIV-1_LAI or HIV-2_ROD in the presence or absence of various concentrations of drugs in 96-well microculture plates and were incubated at 37°C for 7 days. After incubation, 100 μl of the medium was removed from each well, an MTT [3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution (10 μl, 7.5 mg/ml in phosphate-buffered saline) was added to each well in the plate, and the mixture was incubated at 37°C for 2 h. After incubation to dissolve the formazan crystals, 100 μl of acidified isopropanol containing 4% (vol/vol) Triton X-100 was added to each well and the optical density measured in a kinetic microplate reader (VMax; Molecular Devices, Sunnyvale, CA). All assays were performed in duplicate. In some experiments, MT-2 cells were chosen as target cells for the MTT assay, since these cells undergo greater HIV-1-elicited cytopathic effects than do MT-4 cells. To determine the sensitivities of the primary HIV-1 isolates to drugs, PHA-PBMCs (10⁶/ml) were exposed to 50 TCID₅₀ of each primary HIV-1 isolate and cultured in the presence or absence of various concentrations of drugs in 10-fold serial dilutions in 96-well microculture plates. To determine the drug susceptibilities of certain laboratory HIV-1 strains, MT-4 cells were employed as target cells, as previously described, with minor modifications (26, 27). In brief, MT-4 cells (10⁵/ml) were exposed to 100 TCID₅₀ of drug-resistant HIV-1 strains in the presence or absence of various concentrations of drugs and were incubated at 37°C. On day 7 of culture, the supernatants were harvested and the amounts of p24 (capsid [CA]) Gag protein were determined using a fully automated chemiluminescent enzyme immunoassay system (Lumipulse F; Fujirebio, Inc., Tokyo, Japan) (26, 27). The drug concentrations that suppressed the production of p24 Gag protein by 50% (50% effective concentration [EC₅₀]) were determined by a comparison with the p24 production level in a drug-free control cell culture. All assays were performed in duplicate. The EC₅₀ of each combination shown in the current report represents the average value of the data obtained from two to four independently conducted experiments. The PHA-PBMCs were derived from a single donor in each independent experiment. Thus, two to four different healthy donors were recruited to obtain the data.

In vitro selection of protease inhibitor-resistant HIV-1 variants.

MT-4 cells (10⁵/ml) were exposed to HIV-1_NL4-3 (500 TCID₅₀) and cultured in the presence of various HIV-1 protease inhibitors, with the initial concentration being its EC₅₀. Viral replication was monitored by determining the amount of p24 Gag produced by the MT-4 cells. The culture supernatants were harvested on day 7 and were used to infect fresh MT-4 cells for the next round of culture in the presence of increasing concentrations of each drug. When the virus began to propagate in the presence of the drug, the drug concentration was generally increased 2- to 3-fold. Proviral DNA samples obtained from the lysates of infected cells were subjected to nucleotide sequencing. This drug selection procedure was carried out until the drug concentration reached 5 μM, as previously described (28–30). In the experiments conducted to select drug-resistant variants, the MT-4 cells were also exploited as target cells, since HIV-1 in general replicates at greater levels in MT-4 cells than it does in MT-2 cells, as described above.

Determination of nucleotide sequences.

Molecular cloning and a determination of the nucleotide sequences of HIV-1 strains passaged in the presence of anti-HIV-1 agents were performed as previously described (28). In brief, high-molecular-weight DNA was extracted from HIV-1-infected MT-4 cells by using the InstaGene matrix (Bio-Rad Laboratories, Hercules, CA) and was subjected to molecular cloning, followed by sequence determination. The primers used for the first round of PCR with the entire Gag- and protease-encoding regions of the HIV-1 genome were LTR F1 (5′-GAT GCT ACA TAT AAG CAG CTG C-3′) and PR12 (5′-CTC GTG ACA AAT TTC TAC TAA TGC-3′). The first-round PCR mixture consisted of 1 μl of proviral DNA solution, 10 μl of Premix Taq (Ex Taq version; TaKaRa Bio, Inc., Otsu, Japan), and 10 pmol of each of the first PCR primers, in a total volume of 20 μl. The PCR conditions used were an initial 3 min at 95°C, followed by 35 cycles of 40 s at 95°C, 20 s at 55°C, and 2 min at 72°C, with a final 10 min of extension at 72°C. The first-round PCR products (1 μl) were used directly in the second round of PCR with the primers LTR F2 (5′-GAG ACT CTG GTA ACT AGA GAT C-3′) and KSMA2.1 (5′-CCA TCC CGG GCT TTA ATT TTA CTG GTA C-3′) under the PCR conditions of an initial 3 min at 95°C, followed by 35 cycles of 30 s at 95°C, 20 s at 55°C, and 2 min at 72°C, with a final 10 min of extension at 72°C. The second-round PCR products were purified with spin columns (MicroSpin S-400 HR columns; Amersham Biosciences Corp., Piscataway, NJ), cloned directly using pGEM-T Easy vector (Promega, Fitchburg, WI), and subjected to sequencing with a model 3130 automated DNA sequencer (Applied Biosystems, Foster City, CA).

Determination of viral growth kinetics of GRL-0739-resistant HIV-1_NL4-3 variants and wild-type HIV-1_NL4-3.

The GRL-0739-resistant variant at passage 46 was propagated in fresh MT-4 cells without GRL-0739 for 7 days, and aliquoted HIV-1₇₃₉^r_P46 viral stocks were stored at −80°C until use. MT-4 cells (2.4 × 10⁵) were exposed to the HIV-1₇₃₉^r_P46 or a wild-type HIV-1_NL4-3 preparation containing 10 ng/ml p24 in 6-well culture plates for 3 h, and the newly infected MT-4 cells were washed with fresh medium, divided into 3 fractions, and each cultured with or without GRL-0739 (final concentration, 10⁴/ml MT-4 cells; drug concentrations, 0, 0.1, and 1 μM). The amounts of p24 were measured every 2 days for up to 7 days.

Generation of recombinant HIV-1 clones.

To generate HIV-1 clones carrying the desired amino acid substitutions, site-directed mutagenesis was performed with a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), and the amino acid substitution-containing genomic fragments were introduced into pHIV-1_NL4-3Sma. A determination of the nucleotide sequences of the plasmids confirmed that each clone had the desired amino acid substitution but no unintended amino acid substitutions. Each recombinant plasmid was transfected into COS-7 cells with Lipofectamine LTX transfection reagent (Invitrogen, Carlsbad, CA), and the infectious virions thus made were harvested for 72 h after transfection and stored at −80°C until use.

Determination of apparent blood-brain barrier permeability coefficient of GRL-0739 using a novel in vitro model.

A novel in vitro BBB model (BBB kit; PharmaCo-Cell Ltd., Nagasaki, Japan) incorporating a triple culture of rat-derived astrocytes, pericytes, and monkey-derived endothelial cells (31) was used to determine the apparent permeability BBB coefficients (P_app) (cm/s) of GRL-0739, RAL, FTC, MVC, DRV, caffeine, and sucrose.

The BBB kit was kept at −80°C until thawing on the day of the experiments. Nutritional medium was added to both the brain and blood sides of the wells. This solution consists of Dulbecco's modified Eagle's medium (DMEM) F-12 with 10% (vol/vol) FCS, 100 μg/ml heparin, 1.5 ng/ml basic fibroblast growth factor (bFGF), 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodium selenite, 500 nM hydrocortisone, and 50 μg/ml gentamicin. Fresh medium was added 3 h after thawing, according to the manufacturer's instructions, as well as 24 h later. The plates were incubated at 37°C until day 4 of the experiment, when the condition of each astrocyte was checked under a light microscope. Following this, the integrity of the collagen-coated membrane was verified by measuring the transendothelial electrical resistance (TEER) using an ohmmeter. As TEER increases over the days and reaches its peak between days 4 and 6 of the experiment, determinations were made during this period. The membranes were tested individually, and those collagen-coated membranes displaying TEER values of >150 Ω/cm² were suitable for executing the drug BBB penetration assay. Detailed information regarding the components of the BBB kit and its mechanisms can be seen by accessing the manufacturer's website (http://www.pharmacocell.co.jp/en/bbb/index_e.html).

Once the conditions of cell viability and membrane integrity were met, drug dilutions were performed from 20 mM dimethyl sulfoxide (DMSO) stocks of GRL-0739, RAL, FTC, MVC, and DRV, while caffeine and sucrose were used as positive and negative controls, respectively. Standard curves were generated for each compound on a light spectrophotometer, as previously described. A concentration of 100 μM each compound was added to the luminal (blood side) of the wells, wells were incubated at 37°C for 30 min, and the amount of drug that crossed the in vitro BBB was collected and measured under a light spectrophotometer at A₂₃₀.

The P_app value was calculated using the following mathematical formula: P_app (cm/s) = (VA/[A × [C]luminal]) × (Δ[C]abluminal/Δt), where VA is the volume of the abluminal chamber (0.9 cm³), A is the membrane surface area (0.33 cm²), [C]luminal is the initial luminal compound concentration (in μM), Δ[C]abluminal is the abluminal compound concentration (in μM), and Δt is the time of the experiment (in s).

Structural analysis of GRL-0739–protease interaction.

The crystal structures of the GRL-0739–protease (PDB code 4KB9) and DRV-protease (PDB code 2IEN) complexes were obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (http://www.rcsb.org) and analyzed. The highest occupancy conformations of the inhibitors were considered. Hydrogens were added, and the structures were minimized with constraints on heavy atoms with the OPLS-2005 force field, as implemented in Maestro version 9.3 (Schrödinger, LLC, New York, NY). The structural figures were also generated with Maestro. The hydrogen bonds had a distance cutoff of 3.0 Å and angle constraints of 90° with the donor atom and 60° with the acceptor atom.

RESULTS

Antiviral activity of GRL-0739 against HIV-1_LAI and HIV-2_ROD, and their cytotoxicities.

We first examined the antiviral potency of GRL-0739 against a set of HIV-1 isolates. GRL-0739 was effectively active against HIV-1_LAI, with an EC₅₀ of 0.0019 μM compared to other clinically available Food and Drug Administration (FDA)-approved HIV-1 protease inhibitors examined, including DRV (Table 1), as assessed with the MTT assay using MT-2 target cells; however, its cytotoxicity was evident at high concentrations only (50% cytotoxic concentration [CC₅₀], 21.0 μM), and the selectivity index proved to be highly desirable, at 11,053 (Table 1). GRL-0739 was also active against HIV-2_ROD, with an EC₅₀ of 0.0058 μM (Table 1).

TABLE 1.

Antiviral activities of GRL-0739 against HIV-1_LAI and HIV-2_ROD^a

Compound	EC50 (μM)		CC50 (μM)	Selectivity indexb
	HIV-1LAI	HIV-2ROD
GRL-0739	0.0019 ± 0.0007	0.0058 ± 0.0013	21.0 ± 1.6	11,053
SQV	0.028 ± 0.003	0.0032 ± 0.0002	19.1 ± 1.0	682
APV	0.037 ± 0.005	0.34 ± 0.05	60.5 ± 9.6	1,635
ATV	0.0044 ± 0.0006	0.0076 ± 0.0001	26.7 ± 1.3	6,068
DRV	0.0046 ± 0.0006	0.0085 ± 0.0005	137.6 ± 11.0	29,913

^aMT-2 cells (10⁴/ml) were exposed to 100 50% tissue culture infective doses (TCID₅₀) of HIV-1_LAI or HIV-2_ROD and cultured in the presence of various concentrations of each protease inhibitor (PI), and the 50% effective concentration (EC₅₀) values were determined using the MTT assay. All assays were conducted in duplicate, and the data shown represent the mean values ± SD values derived from the results of three independent experiments.

^bEach selectivity index denotes a ratio of the 50% cytotoxic concentration (CC₅₀) to the EC₅₀ against HIV-1_LAI.

GRL-0739 exerts strong activity against highly protease inhibitor-resistant clinical HIV-1 isolates.

In our previous work, we isolated highly multi-protease inhibitor-resistant primary HIV-1 strains, HIV-1_MDR/B, HIV-1_MDR/C, HIV-1_MDR/G, HIV-1_MDR/TM, HIV-1_MDR/MM, and HIV-1_MDR/JSL, from patients with AIDS who had failed anti-HIV regimens after receiving 9 to 11 anti-HIV-1 drugs over 32 to 83 months (23, 24). These primary strains contained 9 to 14 amino acid substitutions in the protease-encoding region, which reportedly are associated with HIV-1 resistance against various protease inhibitors (Table 2, footnotes). The six different multidrug-resistant clinical isolates used in the assays, shown in Table 2, contained various resistance-associated amino acid mutations in the reverse transcriptase (RT) and in the protease. All patients from whom these variants were isolated had received 6 different nucleoside reverse transcriptase inhibitors (NRTIs), and 2 patients had received 1 nonnucleoside reverse transcriptase inhibitor (NNRTI). The potencies of APV, ATV, and LPV against such clinical multidrug-resistant HIV-1 strains were significantly compromised, as examined in PHA-PBMCs as target cells, using p24 production inhibition as an endpoint (Table 2). However, GRL-0739 exerted strong antiviral activity, and its EC₅₀s against those clinical variants were substantially low, at 0.007 to ∼0.033 μM (Table 2). The antiviral activity of GRL-0739 proved to be most effective against those multidrug-resistant clinical HIV-1 variants examined compared to the four FDA-approved HIV-1 protease inhibitors (APV, ATV, LPV, and DRV), except for HIV-1_MDR/MM.

TABLE 2.

Antiviral activities of GRL-0739 and other agents against multidrug-resistant clinical isolates in PHA-PBMCs

Virusa	EC50 (μM) forb:
	GRL-0739	APV	ATV	LPV	DRV
HIV-1WT/ERS104pre	0.0036 ± 0.0003	0.028 ± 0.006	0.0025 ± 0.0004	0.030 ± 0.005	0.0039 ± 0.0006
HIV-1MDR/B (X4)	0.025 ± 0.001 (7)	0.50 ± 0.08 (19)	0.442 ± 0.002 (177)	>1 (>33)	0.034 ± 0.009 (9)
HIV-1MDR/C (X4)	0.007 ± 0.004 (2)	0.30 ± 0.02 (11)	0.039 ± 0.003 (17)	0.41 ± 0.04 (14)	0.011 ± 0.002 (3)
HIV-1MDR/G (X4)	0.010 ± 0.007 (3)	0.49 ± 0.07 (18)	0.029 ± 0.007 (12)	0.17 ± 0.02 (7)	0.025 ± 0.008 (6)
HIV-1MDR/TM (X4)	0.012 ± 0.007 (3)	0.48 ± 0.01 (17)	0.074 ± 0.003 (30)	0.43 ± 0.06 (14)	0.026 ± 0.007 (7)
HIV-1MDR/MM (R5)	0.033 ± 0.009 (9)	0.34 ± 0.02 (12)	0.21 ± 0.02 (84)	0.57 ± 0.10 (19)	0.019 ± 0.002 (5)
HIV-1MDR/JSL (R5)	0.027 ± 0.09 (8)	0.49 ± 0.01 (18)	0.27 ± 0.07 (108)	>1 (>33)	0.028 ± 0.007 (7)

^aAmino acid substitutions identified in the protease-encoding region compared to the consensus type B sequence cited from the Los Alamos database include L63P in HIV-1_ERS104pre; L10I, K14R, L33I, M36I, M46I, F53I, K55R, I62V, L63P, A71V, G73S, V82A, L90M, and I93L in HIV-1_MDR/B; L10I, I15V, K20R, L24I, M36I, M46L, I54V, I62V, L63P, K70Q, V82A, and L89M in HIV-1_MDR/C; L10I, V11I, T12E, I15V, L19I, R41K, M46L, L63P, A71T, V82A, and L90M in HIV-1_MDR/G; L10I, K14R, R41K, M46L, I54V, L63P, A71V, V82A, L90M, and I93L in HIV-1_MDR/TM; L10I, K43T, M46L, I54V, L63P, A71V, V82A, L90M, and Q92K in HIV-1_MDR/MM; and L10I, L24I, I33F, E35D, M36I, N37S, M46L, I54V, R57K, I62V, L63P, A71V, G73S, and V82A in HIV-1_MDR/JSL. HIV-1_ERS104pre served as a source of wild-type HIV-1. X4 and R5 denote the X4-tropic HIV-1 strain and R5-tropic HIV-1 strain, respectively.

^bThe 50% effective concentration (EC₅₀) values were determined by using PHA-PBMCs as target cells, and the inhibition of p24 Gag protein production by each drug was used as an endpoint. The numbers in parentheses represent the fold changes in the EC₅₀s for each isolate compared to the EC₅₀s for HIV-1_ERS104pre. All assays were conducted in duplicate, and the data shown represent the mean values ± SD values derived from the results of two to four independent experiments. The PHA-PBMCs were derived from a single donor in each independent experiment.

GRL-0739 is active against various protease inhibitor-selected laboratory HIV-1 variants.

We also examined GRL-0739 against an array of HIV-1_NL4-3 variants, which had been selected by propagating HIV-1_NL4-3 in the presence of increasing concentrations (up to 5 μM) of each of 3 FDA-approved HIV-1 protease inhibitors (RTV, APV, and ATV) in MT-4 cells (18, 28). Such variants had acquired various HIV-1 protease inhibitor resistance-associated amino acid substitutions in the protease-encoding region of the viral genome (Table 3, footnotes). Each variant was highly resistant to the HIV-1 protease inhibitor by which the variant was selected and showed significant resistance, with an EC₅₀ of >1 μM. GRL-0739 was active against all the variants (except for HIV-1_APV^r_{5 μM}), with EC₅₀s of 0.035 to ∼0.058 μM (the fold differences were 10 to 17 compared to those against HIV-1_NL4-3) (Table 3). Overall, GRL-0739 generally exerted stronger antiviral activity against various wild-type HIV-1 strains, drug resistance variants, and the HIV-2 strain than those of other conventional HIV-1 protease inhibitors (Tables 1 to 3).

TABLE 3.

Antiviral activities of GRL-0739 and other agents against laboratory PI-resistant HIV-1 variants

Virusa	EC50 (μM) forb:
	GRL-0739	APV	ATV	LPV	DRV
HIV-1NL4-3	0.0034 ± 0.0009	0.023 ± 0.002	0.0036 ± 0.0006	0.035 ± 0.001	0.0035 ± 0.0004
HIV-1RTVr5 μM	0.058 ± 0.008 (17)	0.53 ± 0.04 (23)	0.046 ± 0.008 (13)	0.60 ± 0.09 (17)	0.031 ± 0.001 (9)
HIV-1APVr5 μM	0.31 ± 0.02 (91)	>1 (>43)	0.38 ± 0.01 (106)	>1 (>29)	0.46 ± 0.03 (131)
HIV-1ATVr5 μM	0.035 ± 0.001 (10)	0.48 ± 0.14 (21)	>1 (>278)	>1 (>29)	0.036 ± 0.005 (10)

^aThe amino acid substitutions identified in the protease-encoding region compared to the wild-type HIV-1_NL4-3 include M46I, V82F, and I84V in HIV-1_RTV^r_{5 μM}; L10F, V32I, M46I, I54M, A71V, and I84V in HIV-1_APV^r_{5 μM}; and L23I, E34Q, K43I, M46I, I50L, G51A, L63P, A71V, V82A, and T91A in HIV-1_ATV^r_{5 μM}.

^bThe 50% effective concentration (EC₅₀) values were determined by using MT-4 cells as target cells. MT-4 cells (10⁵/ml) were exposed to 100 TCID₅₀ of each HIV-1 virus, and the inhibition of p24 Gag protein production by each drug was used as an endpoint. The numbers in parentheses represent the fold changes in the EC₅₀s for each isolate compared to the EC₅₀s for HIV-1_NL4-3. All assays were conducted in duplicate, and the data shown represent the mean values ± SD values derived from the results of two to four independent experiments.

Effects of human serum proteins on the antiretroviral activity of GRL-0739.

The binding of human serum proteins to a drug is an important determinant of its pharmacological activity in vivo, because overly tight binding may result in a reduction in the interactions between the drug and its target (32). We thus determined the effects of the binding of human serum albumin (HSA) and α₁-acid glycoprotein (AAG) on the antiretroviral activity of GRL-0739 in vitro. Physiologically normal concentrations of HSA (40 mg/ml) and AAG (10 μM) were used to evaluate their binding effects on the activity of GRL-0739 against a wild-type clinical isolate, HIV-1_ERS104pre. All four FDA-approved drugs substantially maintained their activities in the presence of HSA, with a reduction in activity by up to 6-fold relative to the activity in the absence of additional HSA (Table 4). The activities of those HIV-1 protease inhibitors were reduced in the presence of AAG by 9- to 12-fold (Table 4). However, the binding effects of both HSA and AAG on the activity of GRL-0739 were insignificant, with no difference or only a 6-fold difference. Of note, the absolute EC₅₀s of GRL-0739 with HSA or AAG (0.004 to ∼0.020 μM) were better than those of the four FDA-approved HIV-1 protease inhibitors (Table 4).

TABLE 4.

Nonspecific binding effects of human serum proteins on antiviral activity of GRL-0739 and other agents^a

Compound	EC50 (μM) against HIV-1ERS104pre
	+10% FCS	+HSA	+AAG
GRL-0739	0.0036 ± 0.0004	0.004 ± 0.001 (1)	0.020 ± 0.006 (6)
APV	0.028 ± 0.006	0.04 ± 0.02 (1)	0.29 ± 0.01 (10)
ATV	0.0025 ± 0.0004	0.015 ± 0.002 (6)	0.03 ± 0.01 (12)
LPV	0.030 ± 0.005	0.034 ± 0.003 (1)	0.26 ± 0.01 (9)
DRV	0.0039 ± 0.0006	0.008 ± 0.001 (2)	0.038 ± 0.003 (10)

^aHuman serum albumin (HSA) at 40 mg/ml or α₁-acid glycoprotein (AAG) at 10 μM was used to evaluate the binding effects of human serum proteins on the antiviral activity of GRL-0739. The EC₅₀s against HIV-1_ERS104pre with or without HSA or AAG were determined by a p24 assay using PHA-PBMCs as target cells, and the inhibition of p24 Gag production by each drug was used as an endpoint. The numbers in parentheses represent the fold changes in the 50% effective concentration (EC₅₀) values compared to the values without HSA or AAG. The data shown represent the mean values ± SD values derived from the results of two or three independent experiments. The PBMCs were derived from a single donor in each independent experiment.

In vitro selection of HIV-1 variants resistant to GRL-0739.

We next attempted to select HIV-1 variants resistant to GRL-0739 by propagating a laboratory HIV-1 strain, HIV-1_NL4-3, in MT-4 cells in the presence of increasing concentrations of GRL-0739, as previously described (18). HIV-1_NL4-3 was initially exposed to 0.003 μM GRL-0739 and underwent 46 passages to be found to have acquired a 1,667-fold increase (5 μM) in the concentration of GRL-0739 compared to that at the initiation of the selection. Judging from the amounts of p24 Gag protein produced in the culture medium (up to ∼315 ng/ml), the replicative capacity of HIV-1_NL4-3 at passage 46 (HIV₇₃₉^r_P46) was thought to have been reasonably well maintained. Compared to the kinetics of the emergence of variants that are resistant to APV, the emergence of GRL-0739-resistant variants was substantially delayed (Fig. 2). The protease-encoding region of the proviral DNA isolated from the infected MT-4 cells was cloned and sequenced at passages 5, 12, 19, 27, 36, and 46 under GRL-0739 selection. The sequences of the cloned regions and the frequency (%) of identical sequences at each passage are depicted in Fig. 3. By passage 5, the wild-type protease gene sequence was seen in 15 of 17 clones. However, by passage 12, the virus had mostly acquired the L10F, I47V, and I84V substitutions. As the passages proceeded, greater numbers of amino acid substitutions emerged. At passage 27, the M46I substitution was seen in all clones, and the V82I substitution became dominant (in 36 of 40 clones). The L97I substitution was also observed in 14 of the 40 clones at passage 27. However, the L97I substitution disappeared by passage 36. At passage 36, the L19V substitution was observed in 16 of the 25 clones and became dominant at the following passage. At passage 46, the virus had acquired 7 dominant substitutions, including L63P. Interestingly, the I84V substitution changed to the I84A substitution by passage 46 (Fig. 3). The locations of these amino acid substitutions in the protease (PR) dimer are illustrated in Fig. 4. We also examined the effects of each of the 4 substitutions that emerged during the GRL-0739 selection experiment (L10F, M46I, I47V, and V82I) (see Table S1 in the supplemental material).

FIG 2.

In vitro selection of HIV-1 protease inhibitor-resistant HIV-1 variants. HIV-1_NL4-3 was propagated in MT-4 cells in the presence of increasing concentrations of amprenavir (●) or GRL-0739 (○). Each passage of virus was conducted in a cell-free manner. The selection data of the amprenavir-resistant variant were previously reported in reference 17. wk, weeks.

FIG 3.

Amino acid sequences of the protease-encoding region of HIV-1_NL4-3 variants selected in the presence of GRL-0739. The amino acid sequences of protease, deduced from the nucleotide sequence of the protease-encoding region of each proviral DNA isolated at each indicated time, are shown. The amino acid sequence of the wild-type HIV-1_NL4-3 protease is illustrated at the top as a reference. The number of each clone identified over the number of all the clones generated and sequenced is shown on the far right.

FIG 4.

Locations of amino acid substitutions identified as selected in vitro in the presence of increasing concentrations of GRL-0739. The substituted residues and GRL-0739 are shown.

HIV₇₃₉^r_P46 can grow at a high concentration of GRL-0739.

Since the viral growth kinetics of HIV₇₃₉^r_P46 was thought to be reasonably well maintained despite the presence of GRL-0739 mentioned above, we determined the viral growth kinetics of HIV₇₃₉^r_P46 and HIV-1_NL4-3. As shown in Fig. 5A, HIV-1_NL4-3 failed to grow in the presence of GRL-0739 at concentrations as low as 0.1 μM during the entire culture period of 7 days. However, HIV₇₃₉^r_P46 was capable of growth in the presence of 0.1 and 1 μM GRL-0739, and the amount of p24 produced in the culture medium reached the amount without GRL-0739 by day 7 (Fig. 5B). We also determined the antiviral activities of various protease inhibitors (PIs) plus raltegravir against an HIV-1 strain selected with GRL-0739 over 46 passages (HIV-1₇₃₉^r_P46). The data are shown in Table S2 in the supplemental material. Interestingly, HIV-1₇₃₉^r_P46 turned out to be resistant to GRL-0739 (fold difference, >294), while the fold differences with SQV, LPV, and DRV were much smaller than that with GRL-0739.

FIG 5.

Viral growth kinetics of HIV-1_NL4-3 and HIV₇₃₉^r_P46. MT-4 cells (2.4 × 10⁵) were exposed to an HIV-1_NL4-3 (A) or HIV-1₇₃₉^r_P46 (B) preparation containing 10 ng/ml p24 in 6-well culture plates for 3 h, and those MT-4 cells were washed with fresh medium and divided into 3 fractions, and each was cultured with or without GRL-0739 (final concentration of MT-4 cells, 10⁴/ml; drug concentrations, 0, 0.1, and 1 μM). The amount of p24 in each culture flask was measured every 2 days for up to 7 days once in each time point. Sup. p24 (ng/ml) denotes the amounts of p24 Gag protein secreted into culture medium. All p24 values are single-point determinations.

GRL-0739 penetrates well across the blood-brain barrier, as tested in an in vitro assay.

We also attempted to evaluate whether GRL-0739 had an optimal apparent permeability blood-brain barrier (BBB) coefficient by employing an in vitro model using a triple-cell coculture system with rat astrocytes, pericytes, and monkey endothelial cells. This model (BBB kit) is thought to represent an in vitro BBB model for drug transport assays, permitting an adequate cross talk of the cell lines involved and providing a way to test an apparent passage of small molecules across the BBB, as previously described by Nakagawa et al. (33). RAL, FTC, MVC, DRV, or GRL-0739 was added to the luminal interface (blood side) of microtiter culture wells under the optimal conditions for transendothelial electrical resistance (TEER) determination. The concentrations of each compound that permeated into the abluminal interface (brain side) were determined using a spectrophotometer 30 min after the addition of each drug to the blood side of the wells. As shown in Table 5, the amounts of caffeine and sucrose, serving as the most and least lipophilic substances, respectively, in the abluminal interface were 5.13 and 0.05 μM, respectively. Six conventional anti-HIV-1 drugs, RAL, FTC, MVC, LPV, ATV, and DRV, were also used as controls in the assay, in the amounts of 0.68, 1.1, 1.06, 0.94, 1.02, and 0.65 μM, respectively. Unexpectedly, GRL-0739 yielded the greatest concentration (1.8 μM) in the abluminal interface of the microtiter culture wells among the compounds we tested.

TABLE 5.

Determination of apparent BBB permeability coefficients of GRL-0739 and other agents using a novel in vitro model^a

Compound	Class	Initial luminal tracer concn (μM)	Final abluminal tracer concn (μM)b	Papp (10−6 cm/s)b
RAL	Integrase inhibitor	100	0.68 ± 0.23	10.2 ± 3.5
FTC	NRTI	100	1.11 ± 0.44	16.6 ± 6.7
MVC	CCR5 inhibitor	100	1.06 ± 0.41	16.6 ± 6.3
LPV	PI	100	0.94 ± 0.05c	14.2 ± 0.7c
ATV	PI	100	1.02 ± 0.10c	15.4 ± 1.4c
DRV	PI	100	0.65 ± 0.23c	9.9 ± 4.2c
GRL-0739	PI	100	1.80 ± 0.66	27.3 ± 10.1
Caffeine (positive control)		100	5.13 ± 0.28	77.7 ± 4.2
Sucrose (negative control)		100	0.05	0.76

^aIn the in vitro model using a triple coculture of rat astrocytes, pericytes, and monkey endothelial cells, RAL, FTC, MVC, DRV, GRL-0739 (all 100 μM), and the positive and negative controls (caffeine and sucrose, respectively) were added to the luminal interface (termed the blood side) of duplicate wells. The mathematical formula used to calculate P_app is described in Materials and Methods.

^bThe results show the mean values ± SD values from duplicated determinations.

^cData were previously reported in reference 19.

The apparent permeability coefficient (P_app), referred to as a brain uptake index (BUI), is a way to quantitatively and qualitatively determine the penetration efficiency of a compound across a BBB model (34). The P_app value of GRL-0739 (27.3 × 10⁻⁶ cm/s) was greater than that of DRV (9.9 × 10⁻⁶ cm/s) and those of other antiviral drugs tested: RAL, 10.2 × 10⁻⁶ cm/s; FTC, 16.6 × 10⁻⁶ cm/s; MVC, 16.0 × 10⁻⁶ cm/s; LPV, 14.2 × 10⁻⁶ cm/s; and ATV, 15.4 × 10⁻⁶ cm/s. The compounds with apparent permeability coefficients of >20 × 10⁻⁶ cm/s are thought to have reasonably efficient penetration across the BBB, those with values of 10 × 10⁻⁶ to 20 × 10⁻⁶ cm/s have a moderate degree of penetration, and those with values of <10 × 10⁻⁶ cm/s do not penetrate the BBB well (33).

GRL-0739 forms greater van der Waals contacts with protease than does DRV.

Finally, we conducted a structural analysis of the interactions of GRL-0739 with wild-type HIV-1 protease. GRL-0739 was shown to form desirable polar interactions with the backbone atoms of Asp29, Gly27, and Asp30′ and the side chains of two catalytic aspartates, Asp25 and Asp25′ (Fig. 6A). Polar interactions of GRL-0739 with Ile50 and Ile50′ through a bridging water molecule were also identified. Another water molecule was found to mediate hydrogen bond interactions between a tricyclic moiety oxygen of GRL-0739 and the backbone atoms of Asp29 and Gly27 (Fig. 6A). Together, these results strongly suggest that despite the anti-HIV-1 activity of GRL-0739 being comparable to that of DRV, GRL-0739 had much fewer polar interactions with protease than those of DRV (17).

FIG 6.

Molecular interactions of GRL-0739 with HIV-1 protease. (A) Polar interactions of GRL-0739 with HIV-1 protease. GRL-0739 forms polar interactions with the backbone atoms of Gly27, Asp29, Asp30′, and the side chains of catalytic aspartates Asp25 and Asp25′ of HIV-1 protease (shown in green and red ribbons, respectively, with selected amino acid residues in wire representation). The polar interactions of GRL-0739 with Ile50 and Ile50′ through a bridging water molecule are also shown. Another water molecule mediates hydrogen bond interactions between the tricyclic moiety oxygen of GRL-0739 and the backbone atoms of the Asp29 and Gly27 of protease. (B) van der Waals surfaces of protease (shown in gray), GRL-0739 (stick representation, gray carbons), and DRV (stick representation, green carbons). The crystal structure of DRV-protease (PDB ID 2IEN) was superimposed on that of GRL-0739-protease (shown in yellow and orange ribbons). The van der Waals surface of GRL-0739 is shown in red and that of DRV in green. The tricyclic ring of GRL-0739 has greater van der Waals contacts with protease than does DRV. (C) Molecular surface diagrams of GRL-0739 (red surface, gray carbons) and DRV (green surface, green carbons) from the overlay of the corresponding crystal structures. The tricyclic ring of GRL-0739 has a larger surface than does the bis-THF group of DRV. The methoxybenzene of GRL-0739 also has a greater surface than does the aniline of DRV. All figures were generated with Maestro version 9.3, Schrödinger, LLC.

Therefore, the hydrophobic interactions of GRL-739 were additionally examined (Fig. 6B). To this end, the van der Waals surfaces of GRL-0739 and DRV were superimposed for a comparison of the two protease inhibitors. It appeared that the tricyclic ring of GRL-0739 has tighter van der Waals contacts with protease than does DRV (Fig. 6B). When the molecular surface diagrams of GRL-0739 and DRV from the overlay of the corresponding crystal structures were superimposed, the tricyclic ring of GRL-0739 was shown to have a larger surface than that of the bis-THF group of DRV (Fig. 6C). The methoxybenzene of GRL-0739 also had a larger surface than the aniline moiety of DRV. The tricyclic and methoxybenzene groups of GRL-0739 seemed to make greater van der Waals contacts with protease than did the corresponding moieties of DRV (Fig. 6C). These better van der Waals contacts of GRL-0739 might offset the decrease in polar contacts compared to those of DRV and are probably responsible for the comparable potency of GRL-0739 with that of DRV.

DISCUSSION

GRL-0739, which contains a unique cyclic ether-derived nonpeptide P2 ligand, cyclohexyl-bis-tetrahydrofuranylurethane (cyclohexyl-bis-THF), as well as a sulfonamide isostere, suppressed the replication of wild-type HIV-1 and HIV-2, with extremely low EC₅₀s (Table 1). GRL-0739 was highly active against a variety of multidrug-resistant clinical HIV-1 isolates, with EC₅₀s ranging from 0.007 to 0.033 μM, while the existing FDA-approved HIV-1 protease inhibitors examined either failed to suppress the replication of those isolates or required much higher concentrations for viral inhibition (Table 2). GRL-0739 also inhibited the replication of laboratory HIV-1 protease inhibitor-selected HIV-1 variants (except HIV-1_APV^r_{5 μM}), with low EC₅₀s (Table 3). GRL-0739 was relatively less active against HIV-1_APV^r_{5 μM}, with an EC₅₀ of 0.31 μM (91-fold difference), presumably due to the structural resemblance between GRL-0739 and APV, both of which contain a sulfonamide isostere (Fig. 1).

In our study, all the HIV-1 protease inhibitors examined failed to show a significant reduction in antiviral activity with the addition of HSA in culture medium (Table 4). In contrast, the addition of AAG substantially reduced the antiretroviral activities of APV, ATV, LPV, and DRV by >9-fold, and their EC₅₀s increased to 0.29, 0.03, 0.26, and 0.038 μM, respectively. However, the reduction with GRL-0739 was better than that with other conventional HIV-1 protease inhibitors, by 6-fold, and its absolute EC₅₀ was as low as 0.02 μM (Table 4). AAG is an acute-phase protein, and its concentration can increase upon injury, surgery, inflammation, malignancy, or infection, including HIV-1 infection (32, 35). Therefore, this feature of GRL-0739 may represent an advantage for its effective clinical application.

In our HIV-1_NL4-3 selection experiment using GRL-0739, the emergence of GRL-0739-resistant variants was substantially delayed compared to that with APV. Reportedly, APV-resistant HIV-1 variants contain the V32I, I50V, I54L/M, L76V, I84V, and L90M substitutions (36, 37), and such substitutions were also identified in the present study (data not shown). However, except for I84V, none of these APV resistance-associated amino acid substitutions emerged during the GRL-0739 selection (Fig. 3 and 4). It is particularly noteworthy that we failed to select the A28S amino acid substitution in the present selection experiment of GRL-0739. In our previous studies, strong HIV-1 protease inhibitors, such as TMC-126 and GRL-1398, which contain a para-methoxy group in the P2′ site, the A28S amino acid substitution was identified as a resistant variant (23, 29). Intriguingly, when HIV-1_NL4-3 was selected with GRL-0519, which contains the same para-methoxy group in the P2′ site, the A28S substitution never appeared up to passage 37 (18). GRL-0519 has tris-tetrahydrofuranylurethane (tris-THF) as the P2 ligand and the para-methoxy moiety at the P2′ site, suggesting that the presence of tris-THF prevented the selection of the A28S substitution. In this regard, the combination of cyclohexyl-bis-THF as the P2 ligand and para-methoxy moiety at P2′ in GRL-0739 also seems to have prevented the selection of the A28S substitution as a resistant variant. When we generated a series of infectious clones containing each of the amino acid substitutions that emerged upon selection, the fold difference in the antiviral activity of GRL-0739 was not as extensive as we thought, as seen in Table S1 in the supplemental material. Also, when we determined the activity of GRL-0739 against an HIV-1 strain selected with GRL-0739 over 46 passages (HIV-1₇₃₉^r_P46), the fold difference was >294, while the fold difference with DRV tested against HIV-1₇₃₉^r_P46 was only 9 (see Table S2 in the supplemental material), strongly suggesting that the HIV-1 resistance profile is not totally the same between GRL-0739 and DRV.

Zidovudine (ZDV) is the only antiretroviral (ARV) agent with demonstrated efficacy for the treatment of HIV-1-associated dementia (38). Considering that HIV-1 causes CNS diseases ranging from HIV-1-associated neurocognitive disorders (HAND) and the milder, but also serious, presentation, HIV-1-associated minor cognitive/motor disorder (MCMD), to the devastating HIV-1-associated encephalopathy, more effective ARV regimens with agents exerting maximal penetration of the BBB are urgently needed.

Cell culture-based models have greatly contributed to the understanding of the physiology, pathology, and pharmacology of the blood-brain barrier (33). Indeed, certain in vitro BBB models have been proven to serve as useful tools that permit the estimation of the apparent penetration of molecules into the CNS. We have already determined the BBB permeabilities of almost all the currently available anti-HIV-1 drugs (partial data are displayed in Table 5) using in vitro BBB models, and GRL-0739 showed a reasonably desirable index, suggesting effective penetration ability across the BBB compared to that of DRV and other anti-HIV-1 drugs examined in the present study, including RAL, FTC, MVC, LPV, and ATV.

A well-characterized in vitro BBB cell model can also be a valuable tool for studying the mechanistic aspects of transport, as well as the biological and pathological processes related to the BBB (34). To use any in vitro BBB cell model successfully, it needs to fulfill a number of criteria, such as having a reproducible permeability of the reference compounds, good screening capacity, a display of complex tight junctions, an adequate expression of BBB phenotypic transporters, and transcytotic activity. In the present work, GRL-0739 showed the highest P_app values, at 27.3 × 10⁻⁶ cm/s, among the anti-HIV-1 drugs examined (Table 5).

Considering that the EC₅₀s of GRL-0739 are favorably low (Tables 1 to 4) and that the selectivity index of GRL-0739 at 11,053 is considerably better than that of other conventional HIV-1 protease inhibitors examined in this study (Table 1), both the anti-HIV activity and safety of GRL-0739 are desirable, although the efficacy and emergence of adverse effects should ultimately be determined by controlled clinical trials.

As described above, GRL-0739 inhibited the replication of wild-type HIV-1, HIV-2, and various drug-resistant HIV-1 variants with relatively lower concentrations than those of DRV. To clarify the reason for these differences, we performed a structural analysis using the crystal data of the HIV-1 protease-GRL-0739 or -DRV complexes. As illustrated in Fig. 6, the tricyclic ring of GRL-0739 had a greater surface than that of the bis-THF group of DRV. The methoxybenzene of GRL-0739 also had a larger surface than that of the aniline of DRV. Thus, the tricyclic and methoxybenzene groups of GRL-0739 should make greater van der Waals contacts with HIV-1 protease than the corresponding moieties of DRV. These greater van der Waals (hydrophobic) contacts of GRL-0739 should offset the decrease observed in its polar contacts with protease compared to those with DRV (17) and should be responsible for the comparable anti-HIV-1 activities of GRL-0739 and DRV and apparent more desirable CNS penetration profiles of DRV, as assessed using a novel in vitro blood-brain barrier model.

The present data demonstrate that GRL-0739 has desirable features as a candidate drug, with good CNS-penetrating capability for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants; the newly generated cyclohexyl-bis-THF moiety with methoxybenzene should be critical for the strong binding of GRL-0739 to HIV-1 protease, and it should have a good effect as a pharmacophore that confers a high anti-HIV-1 effect in the design of novel protease inhibitors with greater CNS penetration profiles. In conclusion, GRL-0739 possesses a number of desirable features as a candidate drug for treating HIV-1 infection and AIDS, although other various parameters, including oral bioavailability, pharmacokinetics/pharmacodynamics, and biodistribution, remain to be determined, and further investigation is warranted.