Identification of a novel long-acting 4'-modified nucleoside reverse transcriptase inhibitor against HBV.

BACKGROUND & AIMS: While certain nucleos(t)ide reverse transcriptase inhibitors (NRTIs) are efficacious in treating HBV infection, their effects are yet to be optimized and the emergence of NRTI-resistant HBV variants is an issue because of the requirement for lifelong treatment. The development of agents that more profoundly suppress wild-type and drug-resistant HBVs, and that have a long-acting effect, are crucial to improve patient outcomes. METHODS: Herein, we synthesized a novel long-acting 4'-modified NRTI termed E-CFCP. We tested its anti-HBV activity in vitro, before evaluating its anti-HBV activity in HBV-infected human-liver-chimeric mice (PXB-mice). E-CFCP's long-acting features and E-CFCP-triphosphate's interactions with the HBV reverse transcriptase (HBV-RT) were examined. RESULTS: E-CFCP potently blocked HBVWTD1 production (IC50qPCR_cell=1.8 nM) in HepG2.2.15 cells and HBVWTC2 (IC50SB_cell=0.7 nM), entecavir (ETV)-resistant HBVETV-RL180M/S202G/M204V (IC50SB_cell=77.5 nM), and adefovir-resistant HBVADV-RA181T/N236T production (IC50SB_cell=14.1 nM) in Huh7 cells. E-CFCP profoundly inhibited intracellular HBV DNA production to below the detection limit, but ETV and tenofovir alafenamide (TAF) failed to do so. E-CFCP also showed less toxicity than ETV and TAF. E-CFCP better penetrated hepatocytes and was better tri-phosphorylated; E-CFCP-triphosphate persisted intracellularly for longer than ETV-triphosphate. Once-daily peroral E-CFCP administration over 2 weeks (0.02~0.2 mg/kg/day) reduced HBVWTC2-viremia by 2-3 logs in PXB-mice without significant toxicities and the reduction persisted over 1-3 weeks following treatment cessation, suggesting once-weekly dosing capabilities. E-CFCP also reduced HBVETV-RL180M/S202G/M204V-viremia by 2 logs over 2 weeks, while ETV completely failed to reduce HBVETV-RL180M/S202G/M204V-viremia. E-CFCP's 4'-cyano and fluorine interact with both HBVWT-RT and HBVETV-RL180M/S202G-M204 -RT via Van der Waals and polar forces, being important for E-CFCP-triphosphate's interactions and anti-HBV potency. CONCLUSION: E-CFCP represents the first reported potential long-acting NRTI with potent activity against wild-type and treatment-resistant HBV. LAY SUMMARY: Although there are currently effective treatment options for HBV, treatment-resistant variants and the need for lifelong therapy pose a significant challenge. Therefore, the development of new treatment options is crucial to improve outcomes and quality of life. Herein, we report preclinical evidence showing that the anti-HBV agent, E-CFCP, has potent activity against wild-type and treatment-resistant variants. In addition, once-weekly oral dosing may be possible, which is preferrable to the current daily dosing regimens.

Novel Protease Inhibitors Containing C-5-Modified bis-Tetrahydrofuranylurethane and Aminobenzothiazole as P2 and P2' Ligands That Exert Potent Antiviral Activity against Highly Multidrug-Resistant HIV-1 with a High Genetic Barrier against the Emergence of Drug Resistance.

Combination antiretroviral therapy has achieved dramatic reductions in the mortality and morbidity in people with HIV-1 infection. Darunavir (DRV) represents a most efficacious and well-tolerated protease inhibitor (PI) with a high genetic barrier to the emergence of drug-resistant HIV-1. However, highly DRV-resistant variants have been reported in patients receiving long-term DRV-containing regimens. Here, we report three novel HIV-1 PIs (GRL-057-14, GRL-058-14, and GRL-059-14), all of which contain a P2-amino-substituted-bis-tetrahydrofuranylurethane (bis-THF) and a P2'-cyclopropyl-amino-benzothiazole (Cp-Abt). These PIs not only potently inhibit the replication of wild-type HIV-1 (50% effective concentration [EC50], 0.22 nM to 10.4 nM) but also inhibit multi-PI-resistant HIV-1 variants, including highly DRV-resistant HIVDRVRP51 (EC50, 1.6 nM to 30.7 nM). The emergence of HIV-1 variants resistant to the three compounds was much delayed in selection experiments compared to resistance to DRV, using a mixture of 11 highly multi-PI-resistant HIV-1 isolates as a starting HIV-1 population. GRL-057-14 showed the most potent anti-HIV-1 activity and greatest thermal stability with wild-type protease, and potently inhibited HIV-1 protease's proteolytic activity (Ki value, 0.10 nM) among the three PIs. Structural models indicate that the C-5-isopropylamino-bis-THF moiety of GRL-057-14 forms additional polar interactions with the active site of HIV-1 protease. Moreover, GRL-057-14's P1-bis-fluoro-methylbenzene forms strong hydrogen bonding and effective van der Waals interactions. The present data suggest that the combination of C-5-aminoalkyl-bis-THF, P1-bis-fluoro-methylbenzene, and P2'-Cp-Abt confers highly potent activity against wild-type and multi-PI-resistant HIV strains and warrant further development of the three PIs, in particular, that of GRL-057-14, as potential therapeutic for HIV-1 infection and AIDS.

CMCdG, a Novel Nucleoside Analog with Favorable Safety Features, Exerts Potent Activity against Wild-Type and Entecavir-Resistant Hepatitis B Virus.

We designed, synthesized, and characterized a novel nucleoside analog, (1S,3S,5S)-3-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-5-hydroxy-1-(hydroxymethyl)-2-methylene-cyclopentanecarbonitrile, or 4'-cyano-methylenecarbocyclic-2'-deoxyguanosine (CMCdG), and evaluated its anti-hepatitis B virus (anti-HBV) activity, safety, and related features. CMCdG's in vitro activity was determined using quantitative PCR and Southern blotting assays, and its cytotoxicity was determined with a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, while its in vivo activity and safety were determined in human liver-chimeric mice infected with wild-type HBV genotype Ce (HBVWTCe) and an entecavir (ETV)-resistant HBV variant containing the amino acid substitutions L180M, S202G, and M204V (HBVETV-RL180M/S202G/M204V). CMCdG potently inhibited HBV production in HepG2.2.15 cells (50% inhibitory concentration [IC50], ∼30 nM) and HBVWTCe plasmid-transfected Huh7 cells (IC50, 206 nM) and efficiently suppressed ETV-resistant HBVETV-RL180M/S202G/M204V (IC50, 2,657 nM), while it showed no or little cytotoxicity (50% cytotoxic concentration, >500 µM in most hepatocytic cells examined). Two-week peroral administration of CMCdG (1 mg/kg of body weight/day once a day [q.d.]) to HBVWTCe-infected human liver-chimeric mice reduced the level of viremia by ∼2 logs. CMCdG also reduced the level of HBVETV-RL180M/S202G/M204V viremia by ∼1 log in HBVETV-RL180M/S202G/M204V-infected human liver-chimeric mice, while ETV (1 mg/kg/day q.d.) completely failed to reduce the viremia. None of the CMCdG-treated mice had significant drug-related changes in body weights or serum human albumin levels. Structural analyses using homology modeling, semiempirical quantum methods, and molecular dynamics revealed that although ETV triphosphate (TP) forms good van der Waals contacts with L180 and M204 of HBVWTCe reverse transcriptase (RT), its contacts with the M180 substitution are totally lost in the HBVETV-RL180M/S202G/M204V RT complex. However, CMCdG-TP retains good contacts with both the HBVWTCe RT and HBVETV-RL180M/S202G/M204V RT complexes. The present data warrant further studies toward the development of CMCdG as a potential therapeutic for patients infected with drug-resistant HBV and shed light on the further development of more potent and safer anti-HBV agents.

GRL-079, a Novel HIV-1 Protease Inhibitor, Is Extremely Potent against Multidrug-Resistant HIV-1 Variants and Has a High Genetic Barrier against the Emergence of Resistant Variants.

We identified four novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-078, -079, -077, and -058, containing an alkylamine at the C-5 position of P2 tetrahydropyrano-tetrahydrofuran (Tp-THF) and a P2' cyclopropyl (Cp) (or isopropyl)-aminobenzothiazole (Abt) moiety. Their 50% effective concentrations (EC50s) were 2.5 to 30 nM against wild-type HIV-1NL4-3, 0.3 to 6.7 nM against HIV-2EHO, and 0.9 to 90 nM against laboratory-selected PI-resistant HIV-1 and clinical HIV-1 variants resistant to multiple FDA-approved PIs (HIVMDR). GRL-078, -079, -077, and -058 also effectively blocked the replication of HIV-1 variants highly resistant to darunavir (DRV) (HIVDRVrp51), with EC50s of 38, 62, 61, and 90 nM, respectively, while four FDA-approved PIs examined (amprenavir, atazanavir, lopinavir [LPV], and DRV) had virtually no activity (EC50s of >1,000 nM) against HIVDRVrp51 Structurally, GRL-078, -079, and -058 form strong hydrogen bond interactions between Tp-THF modified at C-5 and Asp29/Asp30/Gly48 of wild-type protease, while the P2' Cp-Abt group forms strong hydrogen bonds with Asp30'. The Tp-THF and Cp-Abt moieties also have good nonpolar interactions with protease residues located in the flap region. For selection with LPV and DRV by use of a mixture of 11 HIVMDR strains (HIV11MIX), HIV11MIX became highly resistant to LPV and DRV over 13 to 32 and 32 to 41 weeks, respectively. However, for selection with GRL-079 and GRL-058, HIV11MIX failed to replicate at >0.08 µM and >0.2 µM, respectively. Thermal stability results supported the highly favorable anti-HIV-1 potency of GRL-079 as well as other PIs. The present data strongly suggest that the P2 Tp-THF group modified at C-5 and the P2' Abt group contribute to the potent anti-HIV-1 profiles of the four PIs against HIV-1NL4-3 and a wide spectrum of HIVMDR strains.

Dimerization of HIV-1 protease occurs through two steps relating to the mechanism of protease dimerization inhibition by darunavir.

Dimerization of HIV-1 protease (PR) subunits is an essential process for PR's acquisition of proteolytic activity, which plays a critical role in the maturation of HIV-1. Recombinant wild-type PR (PR(WT)) proved to dimerize, as examined with electrospray ionization mass spectrometry; however, two active site interface PR mutants (PR(T26A) and PR(R87K)) remained monomeric. On the other hand, two termini interface PR mutants (PR(1-C95A) and PR(97/99)) took both monomeric and dimeric forms. Differential scanning fluorimetry indicated that PR(1-C95A) and PR(97/99) dimers were substantially less stable than PR(WT) dimers. These data indicate that intermolecular interactions of two monomers occur first at the active site interface, generating unstable or transient dimers, and interactions at the termini interface subsequently occur, generating stable dimers. Darunavir (DRV), an HIV-1 protease inhibitor, inhibits not only proteolytic activity but also PR dimerization. DRV bound to protease monomers in a one-to-one molar ratio, inhibiting the first step of PR dimerization, whereas conventional protease inhibitors (such as saquinavir) that inhibit enzymatic activity but not dimerization failed to bind to monomers. DRV also bound to mutant PRs containing the transframe region-added PR (TFR-PR(D25N) and TFR-PR(D25N-7AA)), whereas saquinavir did not bind to TFR-PR(D25N) or TFR-PR(D25N-7AA). Notably, DRV failed to bind to mutant PR containing four amino acid substitutions (V32I, L33F, I54M, and I84V) that confer resistance to DRV on HIV-1. To our knowledge, the present report represents the first demonstration of the two-step PR dimerization dynamics and the mechanism of dimerization inhibition by DRV, which should help design further, more potent novel PIs.

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.

UNLABELLED: 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. IMPORTANCE: 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) .

4'-modified nucleoside analogs: potent inhibitors active against entecavir-resistant hepatitis B virus.

UNLABELLED: Certain nucleoside/nucleotide reverse transcriptase (RT) inhibitors (NRTIs) are effective against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). However, both viruses often acquire NRTI resistance, making it crucial to develop more-potent agents that offer profound viral suppression. Here, we report that 4'-C-cyano-2-amino-2'-deoxyadenosine (CAdA) is a novel, highly potent inhibitor of both HBV (half maximal inhibitory concentration [IC50 ] = 0.4 nM) and HIV-1 (IC50 = 0.4 nM). In contrast, the approved anti-HBV NRTI, entecavir (ETV), potently inhibits HBV (IC50 = 0.7 nM), but is much less active against HIV-1 (IC50 = 1,000 nM). Similarly, the highly potent HIV-1 inhibitor, 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA; IC50 = 0.3 nM) is less active against HBV (IC50 = 160 nM). Southern analysis using Huh-7 cells transfected with HBV-containing plasmids demonstrated that CAdA was potent against both wild-type (IC50 = 7.2 nM) and ETV-resistant HBV (IC50 = 69.6 nM for HBVETV-RL180M/S202G/M204V), whereas ETV failed to reduce HBVETV-RL180M/S202G/M204V DNA even at 1 µM. Once-daily peroral administration of CAdA reduced HBVETV-RL180M/S202G/M204V viremia (P = 0.0005) in human-liver-chimeric/ HBVETV-RL180M/S202G/M204V-infected mice, whereas ETV completely failed to reduce HBVETV-RL180M/S202G/M204V viremia. None of the mice had significant drug-related body-weight or serum human-albumin concentration changes. Molecular modeling suggests that a shallower HBV-RT hydrophobic pocket at the polymerase active site can better accommodate the slightly shorter 4'-cyano of CAdA-triphosphate (TP), but not the longer 4'-ethynyl of EFdA-TP. In contrast, the deeper HIV-1-RT pocket can efficiently accommodate the 4'-substitutions of both NRTIs. The ETV-TP's cyclopentyl ring can bind more efficiently at the shallow HBV-RT binding pocket. CONCLUSION: These data provide insights on the structural and functional associations of HBV- and HIV-1-RTs and show that CAdA may offer new therapeutic options for HBV patients.

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.

Insights into the mechanism of inhibition of CXCR4: identification of Piperidinylethanamine analogs as anti-HIV-1 inhibitors.

The cellular entry of HIV-1 into CD4(+) T cells requires ordered interactions of HIV-1 envelope glycoprotein with C-X-C chemokine receptor type 4 (CXCR4) receptors. However, such interactions, which should be critical for rational structure-based discovery of new CXCR4 inhibitors, remain poorly understood. Here we first determined the effects of amino acid substitutions in CXCR4 on HIV-1NL 4 - 3 glycoprotein-elicited fusion events using site-directed mutagenesis-based fusion assays and identified 11 potentially key amino acid substitutions, including D97A and E288A, which caused >30% reductions in fusion. We subsequently carried out a computational search of a screening library containing ∼604,000 compounds, in order to identify potential CXCR4 inhibitors. The computational search used the shape of IT1t, a known CXCR4 inhibitor, as a reference and employed various algorithms, including shape similarity, isomer generation, and docking against a CXCR4 crystal structure. Sixteen small molecules were identified for biological assays based on their high shape similarity to IT1t, and their putative binding modes formed hydrogen bond interactions with the amino acids identified above. Three compounds with piperidinylethanamine cores showed activity and were resynthesized. One molecule, designated CX6, was shown to significantly inhibit fusion elicited by X4 HIV-1NL 4 - 3 glycoprotein (50% inhibitory concentration [IC50], 1.9 µM), to inhibit Ca(2+) flux elicited by stromal cell-derived factor 1α (SDF-1α) (IC50, 92 nM), and to exert anti-HIV-1 activity (IC50, 1.5 µM). Structural modeling demonstrated that CX6 bound to CXCR4 through hydrogen bond interactions with Asp97 and Glu288. Our study suggests that targeting CXCR4 residues important for fusion elicited by HIV-1 envelope glycoprotein should be a useful and feasible approach to identifying novel CXCR4 inhibitors, and it provides important insights into the mechanism by which small-molecule CXCR4 inhibitors exert their anti-HIV-1 activities.

A conserved hydrogen-bonding network of P2 bis-tetrahydrofuran-containing HIV-1 protease inhibitors (PIs) with a protease active-site amino acid backbone aids in their activity against PI-resistant HIV.

In the present study, GRL008, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), and darunavir (DRV), both of which contain a P2-bis-tetrahydrofuranyl urethane (bis-THF) moiety, were found to exert potent antiviral activity (50% effective concentrations [EC50s], 0.029 and 0.002 µM, respectively) against a multidrug-resistant clinical isolate of HIV-1 (HIVA02) compared to ritonavir (RTV; EC50, >1.0 µM) and tipranavir (TPV; EC50, 0.364 µM). Additionally, GRL008 showed potent antiviral activity against an HIV-1 variant selected in the presence of DRV over 20 passages (HIVDRV(R)P20), with a 2.6-fold increase in its EC50 (0.097 µM) compared to its corresponding EC50 (0.038 µM) against wild-type HIV-1NL4-3 (HIVWT). Based on X-ray crystallographic analysis, both GRL008 and DRV showed strong hydrogen bonds (H-bonds) with the backbone-amide nitrogen/carbonyl oxygen atoms of conserved active-site amino acids G27, D29, D30, and D30' of HIVA02 protease (PRA02) and wild-type PR in their corresponding crystal structures, while TPV lacked H-bonds with G27 and D30' due to an absence of polar groups. The P2' thiazolyl moiety of RTV showed two conformations in the crystal structure of the PRA02-RTV complex, one of which showed loss of contacts in the S2' binding pocket of PRA02, supporting RTV's compromised antiviral activity (EC50, >1 µM). Thus, the conserved H-bonding network of P2-bis-THF-containing GRL008 with the backbone of G27, D29, D30, and D30' most likely contributes to its persistently greater antiviral activity against HIVWT, HIVA02, and HIVDRV(R)P20.

GRL-04810 and GRL-05010, difluoride-containing nonpeptidic HIV-1 protease inhibitors (PIs) that inhibit the replication of multi-PI-resistant HIV-1 in vitro and possess favorable lipophilicity that may allow blood-brain barrier penetration.

We designed, synthesized, and identified two novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-04810 and GRL-05010, containing the structure-based designed privileged cyclic ether-derived nonpeptide P2 ligand, bis-tetrahydrofuranylurethane (bis-THF), and a difluoride moiety, both of which are active against the laboratory strain HIV-1LAI (50% effective concentrations [EC50s], 0.0008 and 0.003 µM, respectively) with minimal cytotoxicity (50% cytotoxic concentrations [CC50s], 17.5 and 37.0 µM, respectively, 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 various antiviral regimens. GRL-04810 and GRL-05010 also blocked the infectivity and replication of each of the HIV-1NL4-3 variants selected by up to 5 µM lopinavir (EC50s, 0.03 and 0.03 µM, respectively) and atazanavir (EC50s, 0.02 and 0.04 µM, respectively). Moreover, they were active against darunavir (DRV)-resistant variants (EC50 in 0.03 to 0.034 µM range for GRL-04810 and 0.026 to 0.043 µM for GRL-05010), while DRV had EC50s between 0.02 and 0.174 µM. GRL-04810 had a favorable lipophilicity profile as determined with the partition (log P) and distribution (log D) coefficients 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 × 10(-6) and 61.8 × 10(-6) cm/s, respectively. The present data demonstrate that GRL-04810 and GRL-05010 exert efficient activity against a wide spectrum of HIV-1 variants in vitro and suggest that two fluorine atoms added to their bis-THF moieties may well enhance their penetration across the BBB.

GRL-0519, a novel oxatricyclic ligand-containing nonpeptidic HIV-1 protease inhibitor (PI), potently suppresses replication of a wide spectrum of multi-PI-resistant HIV-1 variants in vitro.

We report that GRL-0519, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing tris-tetrahydrofuranylurethane (tris-THF) and a sulfonamide isostere, is highly potent against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50], 0.0005 to 0.0007 µM) with minimal cytotoxicity (50% cytotoxic concentration [CC50], 44.6 µM). GRL-0519 blocked the infectivity and replication of HIV-1NL4-3 variants selected by up to a 5 µM concentration of ritonavir, lopinavir, or atazanavir (EC50, 0.0028 to 0.0033 µM). 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 amprenavir (APV) and DRV. The effects of nonspecific 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 paramethoxy group effectively fill the S2 and S2' binding pockets, respectively, of the protease. The present 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.

P2' benzene carboxylic acid moiety is associated with decrease in cellular uptake: evaluation of novel nonpeptidic HIV-1 protease inhibitors containing P2 bis-tetrahydrofuran moiety.

GRL007 and GRL008, two structurally related nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) 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 (PR(WT)). Both GRL007 (Ki of 12.7 pM with PR(WT)) and GRL008 (Ki of 8.9 pM) inhibited PR(WT) with high potency in vitro. X-ray crystallographic analysis of PR(WT) 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 PR(WT). 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 (50% effective concentration [EC50] of >1 µM) while GRL008 was highly active (EC50 of 0.04 µM) against wild-type HIV-1. High-performance liquid chromatography (HPLC)/mass spectrometry-based cellular uptake assays showed 8.1- and 84-fold higher intracellular concentrations of GRL008 than 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 a 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 anti-HIV-1 potency, favorable toxicity, and binding profile of GRL008 suggest that further optimization of the P2' moiety may improve its antiretroviral features.

Loss of the protease dimerization inhibition activity of tipranavir (TPV) and its association with the acquisition of resistance to TPV by HIV-1.

Tipranavir (TPV), a protease inhibitor (PI) inhibiting the enzymatic activity and dimerization of HIV-1 protease, exerts potent activity against multi-PI-resistant HIV-1 isolates. When a mixture of 11 multi-PI-resistant (but TPV-sensitive) clinical isolates (HIV(11MIX)), which included HIV(B) and HIV(C), was selected against TPV, HIV(11MIX) rapidly (by 10 passages [HIV(11MIX)(P10)]) acquired high-level TPV resistance and replicated at high concentrations of TPV. HIV(11MIX)(P10) contained various amino acid substitutions, including I54V and V82T. The intermolecular FRET-based HIV-1 expression assay revealed that TPV's dimerization inhibition activity against cloned HIV(B) (cHIV(B)) was substantially compromised. The introduction of I54V/V82T into wild-type cHIV(NL4-3) (cHIV(NL4-3(I54V/V82T))) did not block TPV's dimerization inhibition or confer TPV resistance. However, the introduction of I54V/V82T into cHIV(B) (cHIV(B)(I54V/V82T)) compromised TPV's dimerization inhibition and cHIV(B)(I54V/V82T) proved to be significantly TPV resistant. L24M was responsible for TPV resistance with the cHIV(C) genetic background. The introduction of L24M into cHIV(NL4-3) (cHIV(NL4-3(L24M))) interfered with TPV's dimerization inhibition, while L24M increased HIV-1's susceptibility to TPV with the HIV(NL4-3) genetic background. When selected with TPV, cHIV(NL4-3(I54V/V82T)) most readily developed TPV resistance and acquired E34D, which compromised TPV's dimerization inhibition with the HIV(NL4-3) genetic background. The present data demonstrate that certain amino acid substitutions compromise TPV's dimerization inhibition and confer TPV resistance, although the loss of TPV's dimerization inhibition is not always associated with significantly increased TPV resistance. The findings that TPV's dimerization inhibition is compromised with one or two amino acid substitutions may explain at least in part why the genetic barrier of TPV against HIV-1's development of TPV resistance is relatively low compared to that of darunavir.

Synthesis of novel entecavir analogues having 4'-cyano-6''-fluoromethylenecyclopentene skeletons as an aglycone moiety as highly potent and long-acting anti-hepatitis B virus agent.

Encouraged by our recent findings that 4'-cyano-deoxyguanosine (2), entecavir analogues 4 and 5 are highly potent anti-hepatitis B virus (HBV) agents, we designed and synthesized 6 having a hybridized structure of 4 and 5. The chiral quaternary carbon portion at the 4'-position, which is substituted by cyano- and 5'-hydroxymethyl groups, was stereospecifically constructed by radical-mediated 5-exo-dig mode cyclization of 10. The introduction of the fluorine atom into the 6''-position was achieved by radical-mediated stannylation of sulfide (E)-11 and subsequent electrophilic fluorination of (E)-12. The desired (E)-6 was obtained after the introduction of the guanine base into (E)-18 under Mitsunobu conditions and following global deprotection. The stereoisomer (Z)-6 was also prepared by the same procedure using (Z)-12. Compound (E)-6 showed highly potent anti-HBV activity (EC50 = 1.2 nM) with favorable cytotoxicity (CC50 = 93 µM).

GRL-142 binds to and impairs HIV-1 integrase nuclear localization signal and potently suppresses highly INSTI-resistant HIV-1 variants.

Nuclear localization signal (NLS) of HIV-1 integrase (IN) is implicated in nuclear import of HIV-1 preintegration complex (PIC). Here, we established a multiclass drug-resistant HIV-1 variant (HIVKGD) by consecutively exposing an HIV-1 variant to various antiretroviral agents including IN strand transfer inhibitors (INSTIs). HIVKGD was extremely susceptible to a previously reported HIV-1 protease inhibitor, GRL-142, with IC50 of 130 femtomolar. When cells were exposed to HIVKGD IN-containing recombinant HIV in the presence of GRL-142, significant decrease of unintegrated 2-LTR circular cDNA was observed, suggesting that nuclear import of PIC was severely compromised by GRL-142. X-ray crystallographic analyses revealed that GRL-142 interacts with NLS's putative sequence (DQAEHLK) and sterically blocks the nuclear transport of GRL-142-bound HIVKGD's PIC. Highly INSTI-resistant HIV-1 variants isolated from heavily INSTI-experienced patients proved to be susceptible to GRL-142, suggesting that NLS-targeting agents would serve as salvage therapy agents for highly INSTI-resistant variant-harboring individuals. The data should offer a new modality to block HIV-1 infectivity and replication and shed light on developing NLS inhibitors for AIDS therapy.

Identification of SARS-CoV-2 Mpro inhibitors containing P1' 4-fluorobenzothiazole moiety highly active against SARS-CoV-2.

COVID-19 caused by SARS-CoV-2 has continually been serious threat to public health worldwide. While a few anti-SARS-CoV-2 therapeutics are currently available, their antiviral potency is not sufficient. Here, we identify two orally available 4-fluoro-benzothiazole-containing small molecules, TKB245 and TKB248, which specifically inhibit the enzymatic activity of main protease (Mpro) of SARS-CoV-2 and significantly more potently block the infectivity and replication of various SARS-CoV-2 strains than nirmatrelvir, molnupiravir, and ensitrelvir in cell-based assays employing various target cells. Both compounds also block the replication of Delta and Omicron variants in human-ACE2-knocked-in mice. Native mass spectrometric analysis reveals that both compounds bind to dimer Mpro, apparently promoting Mpro dimerization. X-ray crystallographic analysis shows that both compounds bind to Mpro's active-site cavity, forming a covalent bond with the catalytic amino acid Cys-145 with the 4-fluorine of the benzothiazole moiety pointed to solvent. The data suggest that TKB245 and TKB248 might serve as potential therapeutics for COVID-19 and shed light upon further optimization to develop more potent and safer anti-SARS-CoV-2 therapeutics.

Loss of protease dimerization inhibition activity of darunavir is associated with the acquisition of resistance to darunavir by HIV-1.

Dimerization of HIV protease is essential for the acquisition of protease's proteolytic activity. We previously identified a group of HIV protease dimerization inhibitors, including darunavir (DRV). In the present work, we examine whether loss of DRV's protease dimerization inhibition activity is associated with HIV development of DRV resistance. Single amino acid substitutions, including I3A, L5A, R8A/Q, L24A, T26A, D29N, R87K, T96A, L97A, and F99A, disrupted protease dimerization, as examined using an intermolecular fluorescence resonance energy transfer (FRET)-based HIV expression assay. All recombinant HIV(NL4-3)-based clones with such a protease dimerization-disrupting substitution failed to replicate. A highly DRV-resistant in vitro-selected HIV variant and clinical HIV strains isolated from AIDS patients failing to respond to DRV-containing antiviral regimens typically had the V32I, L33F, I54M, and I84V substitutions in common in protease. None of up to 3 of the 4 substitutions affected DRV's protease dimerization inhibition, which was significantly compromised by the four combined substitutions. Recombinant infectious clones containing up to 3 of the 4 substitutions remained sensitive to DRV, while a clonal HIV variant with all 4 substitutions proved highly resistant to DRV with a 205-fold 50% effective concentration (EC(50)) difference compared to HIV(NL4-3). The present data suggest that the loss of DRV activity to inhibit protease dimerization represents a novel mechanism contributing to HIV resistance to DRV. The finding that 4 substitutions in PR are required for significant loss of DRV's protease dimerization inhibition should at least partially explain the reason DRV has a high genetic barrier against HIV's acquisition of DRV resistance.

CCR5 inhibitors: emergence, success, and challenges.

INTRODUCTION: The discovery of CC-chemokine receptor 5 (CCR5) as a human immunodeficiency virus type 1 (HIV-1) coreceptor opened a new avenue to exploit CCR5 as a potential target for the intervention of HIV-1's cellular entry. AREAS COVERED: Various small-molecule CCR5 inhibitors were identified in the last decade; however, maraviroc (MVC) is the only CCR5 inhibitor currently used in the clinic. Concerns and challenges that exist for wider clinical use of CCR5 inhibitors are discussed. EXPERT OPINION: Although MVC-containing regimens have been recommended for treatment-naïve patients, MVC appears to have been used as one of drugs for salvage therapy rather than for treating drug-naïve patients. This is apparently due to MVC's twice-daily dosing schedule. Another significant disadvantage is that a costly tropism assay must be performed prior to MVC treatment. The access to inexpensive, sensitive, and rapid tropism tests should be made easily available. Only a few novel CCR5 inhibitors are presently in the pipeline. Development of potent and metabolically-stable novel CCR5 inhibitors allowing once-daily dosing regimens is needed. Development of CXCR4 inhibitors should greatly improve the treatment options available to patients infected with X4- and/or dual-tropic HIV-1 strains in combination with a CCR5 inhibitor.