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
50s) 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 FDA-approved 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 recombination-based 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.
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