Halogen bond interactions of novel HIV-1 protease inhibitors (PI) (GRL-001-15 and GRL-003-15) with the flap of protease are critical for their potent activity against wild-type HIV-1 and multi-pi-resistant variants

We generated two novel nonpeptidic HIV-1 protease inhibitors (PIs), GRL-001-15 and GRL-003-15, which contain unique crown-like tetrahydropyranofuran (Crn-THF) and P2=-cyclopropyl-aminobenzothiazole (Cp-Abt) moieties as P2 and P2= ligands, respectively. GRL-001-15 and GRL-003-15 have meta-monofluorophenyl and para-monofluorophenyl at the P1 site, respectively, exert highly potent activity against wild-type HIV-1 with 50% effective concentrations (EC₅₀s) of 57 and 50 pM, respectively, and have favorable cytotoxicity profiles with 50% cytotoxic concentrations (CC₅₀s) of 38 and 11 M, respectively. The activity of GRL-001-15 against multi-PI-resistant HIV-1 variants was generally greater than that of GRL-003-15. The EC₅₀ of GRL-001-15 against an HIV-1 variant that was highly resistant to multiple PIs, including darunavir (DRV) (HIV-1_DRV^R_P30), was 0.17 nM, and that of GRL-003-15 was 3.3 nM, while DRV was much less active, with an EC₅₀ of 216 nM. The emergence of HIV-1 variants resistant to GRL-001-15 and GRL-003-15 was significantly delayed compared to that of variants resistant to selected PIs, including DRV. Structural analyses of wild-type protease (PR^WT) complexed with the novel PIs revealed that GRL-001-15’s meta-fluorine atom forms halogen bond interactions (2.9 and 3.0 Å) with Gly49 and Ile50, respectively, of the protease flap region and with Pro81= (2.7 and 3.2 Å), which is located close to the protease active site, and that two fluorine atoms of GRL-142-13 form multiple halogen bond interactions with Gly49, Ile50, Pro81=, Ile82=, and Arg8=. In contrast, GRL-003-15 forms halogen bond interactions with Pro81= alone, suggesting that the reduced antiviral activity of GRL-003-15 is due to the loss of the interactions with the flap region.