Multidrug-resistant HIV-1 Variants Research Articles

Design, Synthesis and X-Ray Structural Studies of Potent HIV-1 Protease Inhibitors Containing C-4 Substituted Tricyclic Hexahydro-Furofuran Derivatives as P2 Ligands.

The design, synthesis, X-ray structural, and biological evaluation of a series of highly potent HIV-1 protease inhibitors are reported herein. These inhibitors incorporate novel cyclohexane-fused tricyclic bis-tetrahydrofuran as P2 ligands in combination with a variety of P1 and P2' ligands. The inhibitor with a difluoromethylphenyl P1 ligand and a cyclopropylaminobenzothiazole P2' ligand exhibited the most potent antiviral activity. Also, it maintained potent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The corresponding inhibitor with an enantiomeric ligand was significantly less potent in these antiviral assays. The new P2 ligands were synthesized in optically active form using enzymatic desymmetrization of meso-diols as the key step. To obtain molecular insight, two high-resolution X-ray structures of inhibitor-bound HIV-1 protease were determined and structural analyses have been highlighted.

Beyond darunavir: recent development of next generation HIV-1 protease inhibitors to combat drug resistance.

We report our recent development of a conceptually new generation of exceptionally potent non-peptidic HIV-1 protease inhibitors that displayed excellent pharmacological and drug-resistance profiles. Our X-ray structural studies of darunavir and other designed inhibitors from our laboratories led us to create a variety of inhibitors incorporating fused ring polycyclic ethers and aromatic heterocycles to promote hydrogen bonding interactions with the backbone atoms of HIV-1 protease as well as van der Waals interactions with residues in the S2 and S2' subsites. We have also incorporated specific functionalities to enhance van der Waals interactions in the S1 and S1' subsites. The combined effects of these structural templates are critical to the inhibitors' exceptional potency and drug-like properties. We highlight here our molecular design strategies to promote backbone hydrogen bonding interactions to combat drug-resistance and specific design of polycyclic ether templates to mimic peptide-like bonds in the HIV-1 protease active site. Our medicinal chemistry and drug development efforts led to the development of new generation inhibitors significantly improved over darunavir and displaying unprecedented antiviral activity against multidrug-resistant HIV-1 variants.

Structure-Based Design of Highly Potent HIV-1 Protease Inhibitors Containing New Tricyclic Ring P2-Ligands: Design, Synthesis, Biological, and X-ray Structural Studies.

We describe here design, synthesis, and biological evaluation of a series of highly potent HIV-1 protease inhibitors containing stereochemically defined and unprecedented tricyclic furanofuran derivatives as P2 ligands in combination with a variety of sulfonamide derivatives as P2' ligands. These inhibitors were designed to enhance the ligand-backbone binding and van der Waals interactions in the protease active site. A number of inhibitors containing the new P2 ligand, an aminobenzothiazole as the P2' ligand and a difluorophenylmethyl as the P1 ligand, displayed very potent enzyme inhibitory potency and also showed excellent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The tricyclic P2 ligand has been synthesized efficiently in an optically active form using enzymatic desymmetrization of meso-1,2-(dihydroxymethyl)cyclohex-4-ene as the key step. We determined high-resolution X-ray structures of inhibitor-bound HIV-1 protease. These structures revealed extensive interactions with the backbone atoms of HIV-1 protease and provided molecular insights into the binding properties of these new inhibitors.

Design, Synthesis, and X-ray Studies of Potent HIV-1 Protease Inhibitors with P2-Carboxamide Functionalities.

The design, synthesis, biological evaluation, and X-ray structural studies are reported for a series of highly potent HIV-1 protease inhibitors. The inhibitors incorporated stereochemically defined amide-based bicyclic and tricyclic ether derivatives as the P2 ligands with (R)-hydroxyethylaminesulfonamide transition-state isosteres. A number of inhibitors showed excellent HIV-1 protease inhibitory and antiviral activity; however, ligand combination is critical for potency. Inhibitor 4h with a difluorophenylmethyl as the P1 ligand, crown-THF-derived acetamide as the P2 ligand, and a cyclopropylaminobenzothiazole P2'-ligand displayed very potent antiviral activity and maintained excellent antiviral activity against selected multidrug-resistant HIV-1 variants. A high resolution X-ray structure of inhibitor 4h-bound HIV-1 protease provided molecular insight into the binding properties of the new inhibitor.

Design and Synthesis of Potent HIV-1 Protease Inhibitors Containing Bicyclic Oxazolidinone Scaffold as the P2 Ligands: Structure-Activity Studies and Biological and X-ray Structural Studies.

We have designed, synthesized, and evaluated a new class of potent HIV-1 protease inhibitors with novel bicyclic oxazolidinone derivatives as the P2 ligand. We have developed an enantioselective synthesis of these bicyclic oxazolidinones utilizing a key o-iodoxybenzoic acid mediated cyclization. Several inhibitors displayed good to excellent activity toward HIV-1 protease and significant antiviral activity in MT-4 cells. Compound 4k has shown an enzyme Ki of 40 pM and antiviral IC50 of 31 nM. Inhibitors 4k and 4l were evaluated against a panel of highly resistant multidrug-resistant HIV-1 variants, and their fold-changes in antiviral activity were similar to those observed with darunavir. Additionally, two X-ray crystal structures of the related inhibitors 4a and 4e bound to HIV-1 protease were determined at 1.22 and 1.30 Å resolution, respectively, and revealed important interactions in the active site that have not yet been explored.

Design and Synthesis of Highly Potent HIV-1 Protease Inhibitors Containing Tricyclic Fused Ring Systems as Novel P2 Ligands: Structure-Activity Studies, Biological and X-ray Structural Analysis.

The design, synthesis, and biological evaluation of a new class of HIV-1 protease inhibitors containing stereochemically defined fused tricyclic polyethers as the P2 ligands and a variety of sulfonamide derivatives as the P2' ligands are described. A number of ring sizes and various substituent effects were investigated to enhance the ligand-backbone interactions in the protease active site. Inhibitors 5c and 5d containing this unprecedented fused 6-5-5 ring system as the P2 ligand, an aminobenzothiazole as the P2' ligand, and a difluorophenylmethyl as the P1 ligand exhibited exceptional enzyme inhibitory potency and maintained excellent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The umbrella-like P2 ligand for these inhibitors has been synthesized efficiently in an optically active form using a Pauson-Khand cyclization reaction as the key step. The racemic alcohols were resolved efficiently using a lipase catalyzed enzymatic resolution. Two high resolution X-ray structures of inhibitor-bound HIV-1 protease revealed extensive interactions with the backbone atoms of HIV-1 protease and provided molecular insight into the binding properties of these new inhibitors.

Design of Highly Potent, Dual-Acting and Central-Nervous-System-Penetrating HIV-1 Protease Inhibitors with Excellent Potency against Multidrug-Resistant HIV-1 Variants.

Herein we report the design, synthesis, X-ray structural, and biological studies of an exceptionally potent HIV-1 protease inhibitor, compound 5 ((3S,7aS,8S)-hexahydro-4H-3,5-methanofuro[2,3-b]pyran-8-yl ((2S,3R)-4-((2-(cyclopropylamino)-N-isobutylbenzo[d]thiazole)-6-sulfonamido)-1-(3,5-difluorophenyl)-3-hydroxybutan-2-yl)carbamate). Using structure-based design, we incorporated an unprecedented 6-5-5-ring-fused crown-like tetrahydropyranofuran as the P2-ligand, a cyclopropylaminobenzothiazole as the P2'-ligand, and a 3,5-difluorophenylmethyl group as the P1-ligand. The resulting inhibitor 5 exhibited exceptional HIV-1 protease inhibitory and antiviral potency at the picomolar level. Furthermore, it displayed antiviral IC50 values in the picomolar range against a wide panel of highly multidrug-resistant HIV-1 variants. The inhibitor shows an extremely high genetic barrier against the emergence of drug-resistant variants. It also showed extremely potent inhibitory activity toward dimerization as well as favorable central nervous system penetration. We determined a high-resolution X-ray crystal structure of the complex between inhibitor 5 and HIV-1 protease, which provides molecular insight into the unprecedented activity profiles observed.

A Modified P1 Moiety Enhances In Vitro Antiviral Activity against Various Multidrug-Resistant HIV-1 Variants and In Vitro Central Nervous System Penetration Properties of a Novel Nonpeptidic Protease Inhibitor, GRL-10413.

We report here that GRL-10413, a novel nonpeptidic HIV-1 protease inhibitor (PI) containing a modified P1 moiety and a hydroxyethylamine sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50] of 0.00035 to 0.0018 μM), with minimal cytotoxicity (50% cytotoxic concentration [CC50] = 35.7 μM). GRL-10413 blocked the infectivity and replication of HIV-1NL4-3 variants selected by use of atazanavir, lopinavir, or amprenavir (APV) at concentrations of up to 5 μM (EC50 = 0.0021 to 0.0023 μM). GRL-10413 also maintained its strong antiviral activity against multidrug-resistant clinical HIV-1 variants isolated from patients who no longer responded to various antiviral regimens after long-term antiretroviral therapy. The development of resistance against GRL-10413 was significantly delayed compared to that against APV. In addition, GRL-10413 showed favorable central nervous system (CNS) penetration properties as assessed with an in vitro blood-brain barrier (BBB) reconstruction system. Analysis of the crystal structure of HIV-1 protease in complex with GRL-10413 demonstrated that the modified P1 moiety of GRL-10413 has a greater hydrophobic surface area and makes greater van der Waals contacts with active site amino acids of protease than in the case of darunavir. Moreover, the chlorine substituent in the P1 moiety interacts with protease in two distinct configurations. The present data demonstrate that GRL-10413 has desirable features for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants, with favorable CNS penetration capability, and that the newly modified P1 moiety may confer desirable features in designing novel anti-HIV-1 PIs.

C-5-Modified Tetrahydropyrano-Tetrahydofuran-Derived Protease Inhibitors (PIs) Exert Potent Inhibition of the Replication of HIV-1 Variants Highly Resistant to Various PIs, including Darunavir.

We identified three nonpeptidic HIV-1 protease inhibitors (PIs), GRL-015, -085, and -097, containing tetrahydropyrano-tetrahydrofuran (Tp-THF) with a C-5 hydroxyl. The three compounds were potent against a wild-type laboratory HIV-1 strain (HIV-1(WT)), with 50% effective concentrations (EC50s) of 3.0 to 49 nM, and exhibited minimal cytotoxicity, with 50% cytotoxic concentrations (CC50) for GRL-015, -085, and -097 of 80, >100, and >100 μM, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multidrug-resistant HIV-1 variants (HIVMDR). Importantly, darunavir (DRV) was >1,000 times less active against a highly DRV-resistant HIV-1 variant (HIV-1DRV(R) P51); the three compounds remained active against HIV-1DRV(R) P51 with only a 6.8- to 68-fold reduction. Moreover, the emergence of HIV-1 variants resistant to the three compounds was considerably delayed compared to the case of DRV. In particular, HIV-1 variants resistant to GRL-085 and -097 did not emerge even when two different highly DRV-resistant HIV-1 variants were used as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen bond interactions with the backbone atoms of active-site amino acid residues (Asp29 and Asp30) of HIV-1 protease. A strong hydrogen bonding formation between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings indicate that the three compounds warrant further study as possible therapeutic agents for treating individuals harboring wild-type HIV and/or HIVMDR. Darunavir (DRV) inhibits the replication of most existing multidrug-resistant HIV-1 strains and has a high genetic barrier. However, the emergence of highly DRV-resistant HIV-1 strains (HIVDRV(R) ) has recently been observed in vivo and in vitro. Here, we identified three novel HIV-1 protease inhibitors (PIs) containing a tetrahydropyrano-tetrahydrofuran (Tp-THF) moiety with a C-5 hydroxyl (GRL-015, -085, and -097) which potently suppress the replication of HIVDRV(R) . Moreover, the emergence of HIV-1 strains resistant to the three compounds was considerably delayed compared to the case of DRV. The C-5 hydroxyl formed a strong hydrogen bonding interaction with the carbonyl oxygen atom of Gly48 of protease as examined in the structural analyses. Interestingly, a compound with Tp-THF lacking the hydroxyl moiety substantially decreased activity against HIVDRV(R) . The three novel compounds should be further developed as potential drugs for treating individuals harboring wild-type and multi-PI-resistant HIV variants as well as HIVDRV(R) .

Structure-based design of potent HIV-1 protease inhibitors with modified P1-biphenyl ligands: synthesis, biological evaluation, and enzyme-inhibitor X-ray structural studies.

We report the design, synthesis, X-ray structural studies, and biological evaluation of a novel series of HIV-1 protease inhibitors. We designed a variety of functionalized biphenyl derivatives to make enhanced van der Waals interactions in the S1 subsite of HIV-1 protease. These biphenyl derivatives were conveniently synthesized using a Suzuki-Miyaura cross-coupling reaction as the key step. We examined the potential of these functionalized biphenyl-derived P1 ligands in combination with 3-(S)-tetrahydrofuranyl urethane and bis-tetrahydrofuranyl urethane as the P2 ligands. Inhibitor 21e, with a 2-methoxy-1,1'-biphenyl derivative as P1 ligand and bis-THF as the P2 ligand, displayed the most potent enzyme inhibitory and antiviral activity. This inhibitor also exhibited potent activity against a panel of multidrug-resistant HIV-1 variants. A high resolution X-ray crystal structure of related Boc-derivative 17a-bound HIV-1 protease provided important molecular insight into the ligand-binding site interactions of the biphenyl core in the S1 subsite of HIV-1 protease.

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.

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-1NL4-3 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-2ROD 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.

Highly potent HIV-1 protease inhibitors with novel tricyclic P2 ligands: design, synthesis, and protein-ligand X-ray studies.

The design, synthesis, and biological evaluation of a series of HIV-1 protease inhibitors incorporating stereochemically defined fused tricyclic P2 ligands are described. Various substituent effects were investigated to maximize the ligand-binding site interactions in the protease active site. Inhibitors 16a and 16f showed excellent enzyme inhibitory and antiviral activity, although the incorporation of sulfone functionality resulted in a decrease in potency. Both inhibitors 16a and 16f maintained activity against a panel of multidrug resistant HIV-1 variants. A high-resolution X-ray crystal structure of 16a-bound HIV-1 protease revealed important molecular insights into the ligand-binding site interactions, which may account for the inhibitor's potent antiviral activity and excellent resistance profiles.

Substituent effects on P2-cyclopentyltetrahydrofuranyl urethanes: Design, synthesis, and X-ray studies of potent HIV-1 protease inhibitors

The design, synthesis, and biological evaluation of novel C3-substituted cyclopentyltetrahydrofuranyl (Cp-THF)-derived HIV-1 protease inhibitors are described. Various C3-functional groups on the Cp-THF ligand were investigated in order to maximize the ligand-binding site interactions in the flap region of the protease. Inhibitors 3c and 3d have displayed the most potent enzyme inhibitory and antiviral activity. Both inhibitors have maintained impressive activity against a panel of multidrug resistant HIV-1 variants. A high-resolution X-ray crystal structure of 3c-bound HIV-1 protease revealed a number of important molecular insights into the ligand-binding site interactions.

Recent Developments of Peptidomimetic HIV-1 Protease Inhibitors

HIV protease plays a crucial role in the viral life cycle by processing the viral Gag and Gag-Pol polyproteins into structural and functional proteins essential for viral maturation. Inhibition of HIV-1 protease leads to the production of noninfectious virus particles and hence is an important therapeutic target for antiviral therapy in AIDS patients. Among many strategies to combat this disease, highly active antiretroviral therapy (HAART) with HIV protease inhibitors (PIs) in combination with reverse transcriptase inhibitors and fusion inhibitor continues to be the first line treatment for control of HIV infection. However, the rapid emergence of drug-resistant HIV-1 strains and the appearance of cross-resistance are severely limiting the long-term treatment options. Thus, numerous efforts have been made in the design and synthesis of novel protease inhibitors with broad-spectrum activity against multidrug-resistant HIV-1 variants by medicinal chemists. This review will focus on the substrate-based drug design of novel peptidomimetic PIs in recent years since 2006.

Design of HIV-1 protease inhibitors with C3-substituted hexahydrocyclopentafuranyl urethanes as P2-ligands: synthesis, biological evaluation, and protein-ligand X-ray crystal structure.

We report the design, synthesis, biological evaluation, and the X-ray crystal structure of a novel inhibitor bound to the HIV-1 protease. Various C3-functionalized cyclopentanyltetrahydrofurans (Cp-THF) were designed to interact with the flap Gly48 carbonyl or amide NH in the S2-subsite of the HIV-1 protease. We investigated the potential of those functionalized ligands in combination with hydroxyethylsulfonamide isosteres. Inhibitor 26 containing a 3-(R)-hydroxyl group on the Cp-THF core displayed the most potent enzyme inhibitory and antiviral activity. Our studies revealed a preference for the 3-(R)-configuration over the corresponding 3-(S)-derivative. Inhibitor 26 exhibited potent activity against a panel of multidrug-resistant HIV-1 variants. A high resolution X-ray structure of 26-bound HIV-1 protease revealed important molecular insight into the ligand-binding site interactions.

Design and synthesis of potent HIV-1 protease inhibitors incorporating hexahydrofuropyranol-derived high affinity P(2) ligands: structure-activity studies and biological evaluation.

The design, synthesis, and evaluation of a new series of hexahydrofuropyranol-derived HIV-1 protease inhibitors are described. We have designed a stereochemically defined hexahydrofuropyranol-derived urethane as the P2-ligand. The current ligand is designed based upon the X-ray structure of 1a-bound HIV-1 protease. The synthesis of (3aS,4S,7aR)-hexahydro-2H-furo[2,3-b]pyran-4-ol, (-)-7, was carried out in optically active form. Incorporation of this ligand provided inhibitor 35a, which has shown excellent enzyme inhibitory activity and antiviral potency. Our structure-activity studies have indicated that the stereochemistry and the position of oxygens in the ligand are important to the observed potency of the inhibitor. Inhibitor 35a has maintained excellent potency against multidrug-resistant HIV-1 variants. An active site model of 35a was created based upon the X-ray structure of 1b-bound HIV-1 protease. The model offers molecular insights regarding ligand-binding site interactions of the hexahydrofuropyranol-derived novel P2-ligand.

Probing multidrug-resistance and protein-ligand interactions with oxatricyclic designed ligands in HIV-1 protease inhibitors.

We report the design, synthesis, biological evaluation, and X-ray crystallographic analysis of a new class of HIV-1 protease inhibitors. Compound 4 proved to be an extremely potent inhibitor toward various multidrug-resistant HIV-1 variants, representing a near 10-fold improvement over darunavir (DRV). Compound 4 also blocked protease dimerization with at least 10-fold greater potency than DRV.

Design, synthesis, protein-ligand X-ray structure, and biological evaluation of a series of novel macrocyclic human immunodeficiency virus-1 protease inhibitors to combat drug resistance.

The structure-based design, synthesis, and biological evaluation of a series of nonpeptidic macrocyclic HIV protease inhibitors are described. The inhibitors are designed to effectively fill in the hydrophobic pocket in the S1'-S2' subsites and retain all major hydrogen bonding interactions with the protein backbone similar to darunavir (1) or inhibitor 2. The ring size, the effect of methyl substitution, and unsaturation within the macrocyclic ring structure were assessed. In general, cyclic inhibitors were significantly more potent than their acyclic homologues, saturated rings were less active than their unsaturated analogues and a preference for 10- and 13-membered macrocylic rings was revealed. The addition of methyl substituents resulted in a reduction of potency. Both inhibitors 14b and 14c exhibited marked enzyme inhibitory and antiviral activity, and they exerted potent activity against multidrug-resistant HIV-1 variants. Protein-ligand X-ray structures of inhibitors 2 and 14c provided critical molecular insights into the ligand-binding site interactions.

In Vitro Characterization of Multidrug-Resistant HIV-1 Isolates From a Recently Infected Patient Associated With Dual Tropism and Rapid Disease Progression

Multidrug-resistant (MDR) HIV-1 variants are thought to be less fit than wild-type virus. In 2005, we reported a case of transmitted MDR HIV-1 infection associated with dual tropism and rapid clinical progression. Here we report the in vitro characterization of the virus isolates. Replication characteristics of bulk and clonal isolates from this case (MDR-1) were examined and compared with these of a panel of transmitted MDR and wild-type (WT) viruses (MDR-2 approximately 4, WT-1, 2). Infectivity and frequency of infectious virion of propagated isolates were high in MDR-1 biological clones (mean titer, 3.5 x 10(5) TCID50/mL; mean frequency of infectious virion, 1/2444) and its bulk isolate (3.2 x 10(6) TCID50/mL; 1/301) as compared with the other biological clones (7.3 x 10(3) TCID50/mL; 1/21,320). Upslope (log10 p24/mL/d) of viral replication in peripheral blood mononuclear cell culture was much higher in MDR-1 clones (1.30 +/- 0.30: mean +/- SD) than those of MDR-2 approximately 4 (0.75 +/- 0.08) or WT-1, WT-2 clones (0.82 +/- 0.03). The bulk isolate and dual-tropic biological clones from MDR-1 depleted CD4+ T cells very rapidly in vitro compared with the other viruses tested. These findings support the hypothesis that MDR HIV-1 can effectively evolve and compensate not only to retain high-level replication but also to exhibit virulence associated with rapid disease progression.

Darunavir, a conceptually new HIV-1 protease inhibitor for the treatment of drug-resistant HIV

Our structure-based design strategies which specifically target the HIV-1 protease backbone, resulted in a number of exceedingly potent nonpeptidyl inhibitors. One of these inhibitors, darunavir (TMC114), contains a privileged, structure-based designed high-affinity P2 ligand, 3 ( R ) ,3a ( S ) ,6a ( R ) -bis-tetrahydrofuranylurethane (bis-THF). Darunavir has recently been approved for the treatment of HIV/AIDS patients harboring multidrug-resistant HIV-1 variants that do not respond to previously existing HAART regimens.