A novel HIV-1 protease inhibitor, GRL-044, has potent activity against various HIV-1s with an extremely high genetic barrier to the emergence of HIV-1 drug resistance

Abstract

We designed, synthesized, and identified two novel nonpeptidic HIV-1 protease inhibitors (PIs), GRL-037 and GRL-044, containing P2-tetrahydropyrano-tetrahydrofuran (Tp-THF), P1-benzene and P1-methoxybenzene, respectively, and P2'-isopropyl-aminobenzothiazole (Ip-Abt), based on the structure of the prototypic PI, darunavir (DRV). The 50% inhibitory concentrations (IC 50 s) of GRL-037 and GRL-044 against wild-type HIV-1 NL4-3 were 0.042 and 0.0028-0.0033 nM with minimal cytotoxicity profiles compared to the IC 50 values of four most potent FDAapproved PIs, ranging from 2.6 to 70 nM. GRL-044 was also potent against HIV-2 EHO (IC 50 =0.0004 nM) and various PI-resistant HIV-1 variants (IC 50 ranging from 0.065 to 19 nM). In the selection assays we conducted, the emergence of HIV-1 variants resistant to GRL-044 was significantly delayed compared to that against DRV. Thermal stability test using differential scanning fluorimetry employing purified HIV-1 protease (PR) and SYPRO ® Orange showed that both GRL-037 and GRL-044 tightly bound to PR. A28S substitution emerged in the homologous recombinationbased selection assays with GRL-044. Structural analyses showed that the larger size of GRL-044 over DRV, enabling GRL-044 to fit better to the hydrophobic cavity of protease, contributed to the greater potency of GRL-044 against HIV-1. Structural analyses also suggested that the van der Waals surface contact of GRL-044 with A28' appears to be better compared to that of DRV because of the larger surface of Ip-Abt of GRL-044, which may be partially responsible for the emergence of A28S. The present antiviral data and structural features of GRL-044 should provide molecular insights for further design and development of potent and "resistance-repellant" novel PIs.

FMO-guided design of darunavir analogs as HIV-1 protease inhibitors

“…In addition to the H-bond formation with D25′ in the core structure of DRV's F2, the bis -tetrahydrofuranyl (THF) urethane was likely stabilized by A28, D29, D30, I47, and I50′. Designing additional groups, such as tris -THF urethane, methoxy, and gem -difluoro at P2 moiety to introduce H-bonds with the conserved main chain atoms of the PR (D29, D30, G27, and G48), which cannot be easily altered by mutations, was supported by previous findings 73 , 74 . Consequently, our designed substructures were modified by adding hydroxyl (substructures 5 and 6), carboxylate (substructures 1 and 2), and amino groups (substructures 9 to 14) to the bis -THF moiety to increase interactions with additional residues.…”

“…Antiviral DRV analogs (GRL-04410 and GRL-0519) have a methoxy substitution at P2′, as previously described by Weber and co-workers 73 . Modifications with methyl, halogen and isopropyl groups at this position improved the antiviral activity of the analogs 74 , 75 . In our study, to expand hydrogen bonding, hydrophobicity, and electrostatic attraction, we added halogen (such as meta and para-dichloro, para-fluoro in substructures 10, 11, and 12), isopropyl (substructure 18), isopropyl ether (substructure 19), and amino (substructures 1, 2, and 14) groups to the original DRV aromatic ring.…”

Self-Assembled Enzymatic Nanowires with a “Dry and Wet” Interface Improve the Catalytic Performance of Januvia Transaminase in Organic Solvents

“…Differential scanning fluorimetry (DSF) analysis was made to test the thermal stability in terms of denaturation temperature. , The melting temperature ( T m ) and denaturation temperature of free JTA were 48.29 and JTA 54.54 °C, respectively. Compared with free JTA, the melting temperature ( T m ) of JTAnw was 9.02 °C higher, at 57.31 °C, and the denaturation temperature of JTAnw was 11.19 °C higher, at 65.73 °C (Figure a).…”

“…A red laser pointer was employed to identify the Tyndall effect and light scattering from potential precipitated particles by directing a red beam through the solution. 45 Samples were mixed in the assembly buffer and added into disposable cuvettes (12.5 × 12.5 × 45 mm 3 ; As One Labware Group, Osaka, Japan). A red pocket laser pointer (650 nm, max output < 5 mW, No.…”

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

“…1), the substitution appears to be distant from the location of the NLS. However, as in the case of A71V substitution that is distantly located from the catalytic active site of HIV-1 protease and is often seen upon HIV-1's acquisition of resistance to various PIs including DRV (40)(41)(42), the F121V substitution may cause significant alterations of the conformation of the dimer interface so that GRL-142 tightly binds to the NLS. On the other hand, four amino acid substitutions in IN were identified in HIV-1 variants selected with PIs.…”