Development of Antiviral Therapy of HIV-1 Infection

Division Of Clinical Sciences - Nci

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Abstract

Two PIs designated GRL-142 and GRL-121 we have recently reported have been shown to occupy a larger surface in the binding pockets of PR, forming more extensive vdW contacts compared to darunavir. In the present project, we designed and synthesized nine novel PIs that incorporate the unique 6-5-5 ring-fused crown-like tetrahydropyranofuran (Crn-THF) as a P2-ligand as in GRL-142 and GRL-121. Further chemical variations were also introduced with a meta- or para-fluoro-substitution on the benzene ring at the P1 site and replacement of cyclopropyl-aminobenzothiazole (Cp-Abt) of GRL-142 with isopropyl-aminobenzothiazole (Ip-Abt) or isopropyl-aminobenzoxazole (Ip-Abo) as a P2' ligand. We first determined anti-HIV-1 activity of GRL-142 and their derivatives against wild-type HIVNL4-3 (HIVWT) and three DRV-resistant HIV-1 variants (HIVDRVRs; HIVDRVRP20, HIVDRVRP30, and HIVDRVRP51). While early-stage mutations (after 20 weeks of passage) occur mostly in distal region of PR, late-stage mutations (after 30 and 51 weeks of passage) start to appear around the binding pocket. Overall, the nine PIs examined in the present study exerted significantly greater anti-HIV-1 potency (up to 10,000-folds) in cell-based assays and some of them including GRL-001, GRL-002, GRL-003, and GRL-004 proved to have much higher genetic barriers to the emergence of resistant variants as compared to DRV. Generally, the presence of two fluorine atoms in GRL-142 and its congeners (GRL-063 and GRL-016) appears to greatly contribute to their potent anti-HIV-1 activity against HIVWT compared to the activity of GRL-121 that has no fluorine atoms. However, no definite difference was observed between the potency of the mono-meta-fluorine-containing inhibitor group (GRL-001, GRL-002, GRL-014, and GRL-015) and the mono-para-fluorine-containing inhibitor group (GRL-003, GRL-004, and GRL-011). Even though the potency of GRL-063 and GRL-016 was progressively reduced against from HIVDRVRP20 to HIVDRVRP30, and then to HIVDRVRP51 overall, these molecules performed overall much better than mono-fluorine-containing inhibitors. Of particular note, GRL-142 was remarkably potent against the three HIVDRVRs and exerted even greater activity against HIVDRVRP20 and HIVDRVRP30 than against HIVWT as previously reported. Structurally, the antiviral activity was increased when the inhibitors had a cyclopropyl at the P2'-position as compared to an isopropyl together with an aminobenzothiazole moiety. Notably, substitution of P2'-cyclopropyl of GRL-142 with P2'-isopropyl, generating GRL-016, substantially reduced antiviral activity against HIVWT by a factor of Furthermore, the activity of GRL-016 was significantly weakened against HIVDRVRs. We also determined the thermal stability of PRD25N in the presence of DRV, saquinavir (SQV), GRL-001, GRL-002, GRL-003, GRL-004, GRL-063, GRL-121, and GRL-142 using differential scanning fluorimetry (DSF). The melting point/denaturing temperature (Tm) of PRD25N alone was 54.5 degrees; while in the presence of DRV and SQV, the Tm values increased to 57.7 and 57.1 degrees, respectively. However, in the presence of GRL-001 through GRL-142, Tm values of PRD25N dramatically increased. These thermal stability results correlated well with the antiviral activity data, indicating that GRL-142 and its derivatives are much stronger binders to PRD25N and stabilize the structure of PRD25N at higher temperatures than DRV and SQV. Indeed, the Tm values of GRL-002 and GRL-004, both of which were less potent against HIVWT and HIVDRVRs than GRL-001 and GRL-003, were lesser than the Tm values of GRL-001 and GRL-003. The highest Tm value (65.5 degrees) was achieved with GRL-142, suggesting that GRL-142 is a most highly potent binder to PRD25N and that GRL-142 contains a successful fragment, as defined by the increase in melting point by more than 0.8 degrees. We further investigated the effects of fluorination and replacement of the P2'-Ip-Abo moiety on the cell membrane permeability. The permeability across the cell membrane of PBMCs and MT4 cells for GRL-142 and the nine PIs was significantly higher than that of DRV. Especially, two bis-fluorine- and Abt-containing PIs, GRL-142 and GRL-016, showed 88- and 153-times greater cell permeability than DRV, followed by 4 mono-fluorine- and Abt-containing PIs (GRL-001, GRL-003, GRL-011, and GRL-014). No major difference in cell permeability was observed when the fluorine was at the meta- or para-position. All four Abo-containing PIs (GRL-002, GRL-004, GRL-063, and GRL-015) displayed reduced permeability compared to the six Abt-containing PIs mentioned above, independent of mono- or bis-fluorine moiety. Overall, GRL-016 that contains two fluorine atoms at the P1-benzene and P2'-Ip-Abt moiety, displayed the greatest cell permeability, suggesting that the combination of those two moieties is beneficial for permeability. In fact, GRL-011 with Ip-Abt had a 3.2-fold greater permeability than GRL-003 with Cp-Abt, and GRL-014 with Ip-Abt also had 3.4-fold greater permeability than GRL-001 with Cp-Abt, suggesting that Ip-Abt confers higher membrane permeability than Cp-Abt, at least among GRL-142 and its derivatives examined. Our present data demonstrate that fine-tuning the DRV structure, e.g., by replacement of a cyclopropyl with a saturated isopropyl moiety and/or the introduction of a fluorine atom(s), alters the potency against HIV-1. In particular, our data demonstrate that addition of two fluorine atoms greatly boosts the inhibitory properties of the PIs examined and enhances their membrane penetration capability. We have also examined to what extent the position of such fluorines effect their anti-HIV-1 activity. In addition to the effects described, dual fluorine substitutions may alter metabolic stability and cellular toxicity but these parameters were not examined in details in the present study. The percentage of fluorinated therapeutics in the pharmaceutical market has markedly increased over the decades and in 2018, three out of ten approved new drugs contains at least one fluorine atom. Among the nine FDA-approved PIs, however, only TPV contains three fluorines. Tiprinavir is the first non-peptidic PI and therefore exhibits a higher oral bioavailability. So-called fluorine scan increasingly being used for drug development. To this end, our data may further contribute to the use of fluorine substitutions in the future design of novel PIs. Furthermore, the cyclopropyl-containing inhibitors exerted quite robust activity against most of the drug-resistant HIV-1 variants examined here. Replacement of cyclopropyl with isopropyl at the distal part of the inhibitor's P2' moiety results in a reduction in the anti-viral activity against wild-type HIV-1. Our findings are consistent with the recognized positive properties of cyclopropyl substitutions in drugs, such as increased metabolic stability and cell and blood-brain-barrier permeability. To the best of our knowledge, there is no direct comparison between cyclo- and iso-propyl groups in cell membrane permeability; however, we observed a greater membrane permeability of compounds with an isopropyl group in the current study.