Novel Protease Inhibitors Containing C-5-Modifiedbis-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

Abstract

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 P2ʹ-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.

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

“…There is only one difference: both GRL-077 and GRL-058 have C5-modified Tp-THF, while GRL-044 has unmodified (44) Tp-THF, strongly suggesting that the C5-modification significantly weakens the potency against HIV-1 NL4-3 , although the potency of GRL-077 and GRL-058 against various PI-resistant variants including HIV DRV R P10 and HIV DRV R P30 is comparable with that of GRL-044. Determination of thermal stability using the differential scanning fluorimetry employing SYPRO ® orange helps understand the specificity and binding force of protein and its ligand (19,29,31). The T m values of DRV and SQV, which were greater than those without agent and with AZT, were the lowest and those with GRL-037 and GRL-044 were further greater (Figure 3).…”

“…The reason why both HIV 11MIX 037-1-WK40 and HIV 11MIX 044-1-WK40 were not sufficiently replicative in the homologous recombination-based selection assay could be due to the acquisition of A28S substitution. The A28S substitution has been seen in the selection assay of HIV-1 with a few PIs, such as TMC-126 (26), GRL-98065 (27), brecanavir (25), and GRL-1398 (12), and not with any of the currently available FDA-approved PIs except only in a few cases (28,29). Of note, however, the population size of HIV-1 in the present homologous recombinationbased selection assay is assumed to be significantly smaller than the size of HIV-1 variants such as polymorphic amino acid substitution-containing variants that reside in HIV-1-infected individuals, the appearance of mutations is affected by stochastic phenomena.…”

“…We determined the amino acid sequences by deducing from nucleic acid sequences of the protease of HIV 11MIX exposed to APV, DRV, GRL-037, and GRL-044 over 13,29,40, and 40 weeks in experiment 1 (HIV 11MIX APV-1-WK13 , HIV 11MIX DRV-1-WK29 , HIV 11MIX 037-1-WK40 , and HIV 11MIX 044-1-WK40 ) respectively ( Figure 5). The amino acid sequences showed distinctive differences between the former 2 strains and the latter 2 strains.…”

“…It is thought that the emergence of V32I and I84V took place relatively quickly because of the use of HIV 11MIX as a starting virus population, which led to homologous recombination occurring from one isolate to another in the presence of escalating doses of DRV, expediting the emergence of highly DRV-resistant Interestingly, in both HIV 11MIX 037-1-WK40 , and HIV 11MIX 044-1-WK40 , the unique substitution, A28S, known to lead to a significant reduction in protease activity (24) and poor viral fitness (25), had been acquired by passage 40 (highlighted in red in Figure 5), while both resultant variants had not apparently acquired high levels of resistance to GRL-037 or GRL-044 and did not replicate well in the presence of either of the two compounds (Figure 3). Since the A28S substitution has been seen with a few PIs, such as TMC-126 (26), GRL-98065 (27), brecanavir (25), and GRL-1398 (12) and not with any of the currently available FDA-approved PIs except only in a few cases (28,29), we repeated the selection assay with APV, DRV, and GRL-044 ( Figure 3 , Experiment 2). When we determined the amino acid sequence of the protease of HIV 11MIX with direct nucleic acid sequencing after HIV 11MIX was selected in the presence of GRL-044 in the second selection assay, the same A28S substitution was identified by week 27 as highlighted in red at the bottom of Figure 5.…”

Benzothiazoles as potential antiviral agents

“…This compound had an EC 50 ranging from 1.6 nM to 30.7 nM. [70] Ali and collaborators, at the University of Massachusetts Medical School, developed a series of novel PIs having phenyloxazolidinone P2 ligands (35). SAR studies showed that the introduction of polar substituents, such as methylsulfonyl group at the 3-position of the phenyl ring as P2 ligand significantly increased in-vitro antiviral activity.…”