Addressing Antiretroviral Drug Resistance with Host-Targeting Drugs-First Steps towards Developing a Host-Targeting HIV-1 Assembly Inhibitor

Abstract

The concerning increase in HIV-1 resistance argues for prioritizing the development of host-targeting antiviral drugs because such drugs can offer high genetic barriers to the selection of drug-resistant viral variants. Targeting host proteins could also yield drugs that act on viral life cycle events that have proven elusive to inhibition, such as intracellular events of HIV-1 immature capsid assembly. Here, we review small molecule inhibitors identified primarily through HIV-1 self-assembly screens and describe how all act either narrowly post-entry or broadly on early and late events of the HIV-1 life cycle. We propose that a different screening approach could identify compounds that specifically inhibit HIV-1 Gag assembly, as was observed when a potent rabies virus inhibitor was identified using a host-catalyzed rabies assembly screen. As an example of this possibility, we discuss an antiretroviral small molecule recently identified using a screen that recapitulates the host-catalyzed HIV-1 capsid assembly pathway. This chemotype potently blocks HIV-1 replication in T cells by specifically inhibiting immature HIV-1 capsid assembly but fails to select for resistant viral variants over 37 passages, suggesting a host protein target. Development of such small molecules could yield novel host-targeting antiretroviral drugs and provide insight into chronic diseases resulting from dysregulation of host machinery targeted by these drugs.

Keywords: ABCE1; DDX6; Gag; HIV-1 assembly; HIV-1 capsid; RNA granule; antiretroviral; antiviral; drug screen; viral-host interactions.

Figure 2

Each assembly-defective Gag mutant is arrested at a characteristic point in the HIV-1 capsid assembly pathway. Initial studies in the cell-free system and in HIV-1-expressing cells revealed that Gag progresses through a stepwise pathway of multiprotein complexes defined as intermediates in a pathway of assembly based on pulse-chase experiments (reviewed in [47])). Subsequent experiments confirmed the order of intermediates in this pathway by showing that each assembly-defective Gag mutant is arrested at a characteristic point in this pathway. Altogether five different categories of mutants have been identified (Class 1–5), one for each point in the pathway. The difference between Class 3 and Class 4 being that Class 3 mutants are arrested as cytosolic ~80S complexes while Class 4 mutants are arrested as PM-associated ~80S complexes, suggesting that the ~80S assembly intermediate is the complex in which assembling Gag traffics from the cytosol to the PM. Later studies identified some of the host proteins in the assembly intermediates. Relevant references are in the text.

Figure 1

Diagram of the HIV-1 life cycle showing points of inhibition by FDA-approved drugs. Stages of the life cycle are labeled in blue font above the diagram. Events that are inhibited are shown with vertical blockade arrows below and labeled in black font, with an example of one drug in each category labeled in red font. The pink bar indicates late events in the viral life cycle that are not yet inhibited by an FDA approved drug, leaving a large gap in the ARV armamentarium.

Figure 3

The host-catalyzed HIV-1 assembly pathway was developed into a screen for small molecule inhibitors. (A) Schematic showing the host-catalyzed HIV-1 assembly pathway, starting with Gag synthesis and formation of the early ~10S assembly intermediate. The ~80S assembly intermediate is formed when ~10S Gag co-opts a host RNP complex containing ABCE1 and DDX6, two host enzymes that have been shown to facilitate assembly. The co-opted host RNP complex is distinct from but related to P-bodies and stress granules, which are larger host RNP complexes. The ~80S assembly intermediate targets to the plasma membrane where Gag multimerization continues, resulting in sequential formation of the ~150S and ~500S assembly intermediates and the fully assembled ~750S immature capsid. Upon completion of assembly, the host RNP complex is released. Relevant references are in the text. (B) A cell-free protein synthesis and assembly plate screen that recapitulates the host-catalyzed HIV-1 assembly pathway was developed and utilized to identify small molecule inhibitors of the pathway [29]. In this screen, a capture antibody directed against Gag binds Gag monomers, oligomers, and multimers generated in a cell-free assembly reaction. The same anti-Gag antibody is used as a detection antibody that binds to captured oligomers and multimers, but not monomers whose binding site is occupied. The signal produced by the detection antibody is proportional to the amount of anti-Gag binding with a larger fluorescent signal indicating more extensive multimerization. The upper diagram shows the large signal that is produced when the HIV-1 cell-free assembly reaction is carried out in the presence of DMSO, which does not inhibit Gag assembly. The lower diagram shows inhibition of that signal when an inhibitor of Gag assembly is added at the start of the cell-free reaction. Legend in the middle applies to panels (A) and (B).

Figure 4

Small molecules that potently inhibit HIV-1 immature capsid assembly were recently described [29]. Structures of two ARV compounds are shown to the left. Dose–response curves for their inhibition of HIV-1 replication in the MT-2 T cell line are shown to the right, with EC50 and CC50 indicated. (A) PAV-206, is a tetrahydroisoquinolone derivative with excellent drug-like properties and a selectivity index (CC50/EC50) of ~82 in MT-2 cells. Other studies show that PAV-206 has a selectivity index of 48 in PBMCs and inhibits virus production most likely by reducing formation of the late ~500S assembly intermediate [29]. (B) PAV-818 is an analog of PAV-206 that retains ARV activity and contains the following modifications—a biotin tag for colocalization studies (shown in red) and a diazirine group for crosslinking (shown in blue). PAV-818 has a selectivity index (CC50/EC50) of ~32 in MT-2 cells.

Figure 5

The biotinylated antiretroviral analog of PAV-206 colocalizes with three components of assembly intermediates but does not colocalize with two host proteins that are not associated with Gag or assembly intermediates [29]. (A) Schematic of the PLA approach for detecting colocalization of PAV-206 with either Gag or the host proteins ABCE1 and DDX6. 293T cells chronically infected with HIV-1 (293T-HIV) were treated with 10 µM PAV-818 (the biotinylated active compound). PLA was performed on treated cells by incubating with primary antibody pairs (either rabbit anti-biotin with mouse anti-Gag; mouse anti-biotin with rabbit anti-ABCE1; or mouse anti-biotin with rabbit anti-ABCE1) followed by incubation with PLA secondary antibodies (anti-rabbit IgG coupled to [+] PLA oligonucleotide and anti-mouse IgG coupled to [−] PLA oligonucleotide). Addition of other PLA reagents results in connector oligonucleotides annealing to the “+” and “–” oligonucleotides only if the primary antibodies are colocalized (within 40 nm); this in turn leads to the PLA amplification reaction. The addition of an oligonucleotide that recognizes a sequence in the amplified regions and is coupled to a red fluorophore (red star) results in intense spots only at sites where the two antibody targets (biotinylated compound and Gag, or biotinylated compound and host protein) are colocalized in situ. After PLA, IF was performed by adding secondary antibody conjugated to a green fluorophore (green star) to detect any unoccupied Gag or host protein antibody, thus marking Gag- or host-protein expressing cells with low-level green fluorescence. (B) This schematic illustrates a scenario in which the biotinylated compound and host protein are more than 40 nm apart. The connector oligonucleotide will not anneal to the antibody-conjugated [+] and [−] PLA oligonucleotides; thus, little to no amplification will occur and few or no red spots will be observed. (C) Data in the three panels on the left show colocalization by PLA of Gag, ABCE1, and DDX6 with PAV-818, respectively. Data in the two panels on the right show lack of colocalization with two host proteins that are found in P-bodies and stress granules, respectively, G3BP1 and XRN1, but are not known to be associated with Gag or found in assembly intermediates. Additional PLA negative controls, PLA dose–response curves, and quantitation are found in [29].

Figure 6

PAV-206 appears to inhibit HIV-1 replication by reducing formation of the ~500S assembly intermediate, most likely by binding to a host protein in assembly intermediates. The top panel shows that the assembly pathway is unaffected by an inactive control compound. For details of events in this pathway, see the Figure 3A legend. The bottom panel is based on recent findings [29] and shows that addition of PAV-206 or one of its active analogs results in association of the small molecule (red asterisk) with an as yet unidentified host protein in the assembly intermediates, leading to a reduction in the formation of the final ~500S assembly intermediate and little to no virus production.