Antiviral Activity of RPV and DOR Against Known NNRTI-Resistant HIV-1 Mutants
The ability of RPV and DOR to inhibit the replication of WT HIV-1 and drug-resistant mutants was measured using a previously described single-round infection assay. We initially investigated the L100I, K103N, Y181C, Y188L, H221Y, and K103N/Y181C RT mutants. These mutants were chosen because they have been selected in patients and are well distributed around the NNRTI binding pocket.[1,2] In previous work, RPV potently inhibited infection of both the WT and the mutant viruses with IC50 values of 0.24 ± 0.1 nM and <2 nM, respectively. DOR potently inhibited the replication of the WT HIV-1 vector in the single-cycle assay (0.67 ± 0.14 nM) and L100I mutant (1.14 ± 0.2 nM) as shown in Fig. 1A. However, the resistant mutants K103N, Y181C, and H221Y showed modest decreases in susceptibility against DOR (ranging from 2–5 nM), and the K103N/Y181C double mutant exhibited a larger drop in susceptibility (11.3 ± 5.9 nM). DOR also showed a considerable loss of potency versus the resistant mutant Y188L, out of the range of our assay (>100 nM).
Antiviral Activities of RPV and DOR Against Other Common NNRTI-resistant Mutants
We determined the antiviral activities of RPV and DOR against some other known NNRTI-resistant mutants to determine the relative strengths and weaknesses of the 2 compounds. We chose NNRTI-resistant mutants that are known to contribute to virological failure in HIV-infected individuals, or mutants that were selected in cell culture: G190A, G190S, M230L, P236L, L100I/K103N, K103N/P225H, and V106A/G190A/F227L. RPV showed potent antiviral activity against all of the tested mutants (≤2 nM across the entire panel), whereas DOR was less effective (Fig. 1B). HIV-1 variants G190A, P236L, and L100I/K103N were susceptible to DOR (<2 nM), however, the G190S mutant showed a modest reduction in susceptibility (4.6 ± 1.2 nM), whereas the M230L mutant and the K103N/P225H double mutant had substantial decreases in their respective susceptibilities (51.1 ± 6.5 and 25.3 ± 4.5 nM, respectively). In addition, the triple mutant V106A/G190A/F227L was relatively insensitive to DOR (>100 nM). These results suggest that RPV is more broadly effective against many NNRTI-resistant mutants.
Antiviral Activities of RPV and DOR Against Mutants Selected by DOR in Cell Culture
V106A was the major variant selected by DOR treatment of HIV-1 in cultured cells. Some of the viruses selected by DOR in culture contained additional mutations: V106A/F227L, V106A/L234I, and V106A/F227L/L234I. To determine whether there is cross-resistance between RPV and DOR, and to further compare the overall effectiveness of the compounds, RPV and DOR were screened against the following mutants: V106A, L234I, V106A/F227L, V106/L234I, and V106A/F227L/L234I (Fig. 2). All 5 of these mutants were sensitive to RPV (<0.8 nM). As expected, L234I (6.8 ± 2.5 nM) showed a modest reduction in susceptibility to DOR, whereas V106A showed a greater loss of susceptibility (15.6 ± 4 nM). The double and triple mutants V106A/F227L, V106/L234I, and V106A/F227L/L234I all showed a substantial loss of susceptibility to DOR (>100 nM).
Antiviral activities of RPV and DOR against HIV-1 that contains mutations selected by DOR in cell culture. The IC50 values of RPV and DOR against vectors that carry WT RT and mutants selected by DOR in cell culture were measured using a single-round infection assay. Error bars represent the standard deviations of independent experiments (n = 4). The IC50 values of the graph have a maximum value of 100 nM, and to better illustrate the lower IC50 values, the Y-axis was broken twice between 1 and 5 nM and 20 and 95 nM; there are similar breaks in the appropriate bars. The IC50 value of DOR against the V106A/F227L, V106A/L234I, and V106A/F227L/L234I resistant mutants were beyond the point of detection in our single round infection assay (.100 nM). The RPV susceptibility data were also used as a control in experiments testing the efficacy of a series of RPV analogs (21).
Antiviral Activities of RPV and DOR Against Mutants Selected by RPV
The E138K substitution in RT is the most common NNRTI resistance mutation seen in patients who fail RPV-containing regimens. Because many of the commonly used cART regimens include either Lamivudine (3TC) or FTC, some isolates from virological failures also contained the M184V/I mutation. K101E is a mutation that is commonly selected in patients who fail regimens that include ETR, a compound that is structurally related to RPV, and that we selected with RPV in vitro. In addition, we selected HIV-1 RT mutations in cell culture with a closely related RPV analog (unpublished observations): E40K, D67E, V111A, E138K, Y181C, and M230I. As described above, both compounds were tested against M230L (which should be similar to M230I). The effects of Y181C on the susceptibility of the vector to DOR and RPV were discussed earlier. We screened a panel of mutants that included E40K, D67E, K101E, V111A, E138K, M184I, M184V, K101E/M184I, K101E/M184V, E138K/M184I, and E138K/M184V (Fig. 3) to determine whether these mutants are susceptible to DOR. DOR potently inhibited the replication of E40K, K101E, V111A, M184I, M184V, K101E/M184I, K101E/M184V, E138K/M184I, and E138K/M184V (all <3 nM). The E138K mutant showed a modest decrease in susceptibility to DOR (13.9 ± 2.4 nM), whereas D67E variant showed a larger decrease in susceptibility (46 ± 14 nM). Interestingly, the variant K101E was the only RPV-associated single mutant which conferred a small, but measurable decrease in susceptibility to RPV (2.6 ± 1.6 nM), whereas the rest of the mutants selected by RPV remained sensitive in our assay (<1 nM). The ability of RPV to inhibit these E138K/M184I/V RT mutants in tissue culture has been reported previously. RPV has been reported to select for another resistance pathway that involves K101P and Y181I, and K101P/V179I.[13,17] We tested DOR and RPV against these resistant mutants (Fig. 4); K101P HIV-1 showed a slight increase in susceptibility to DOR (1.0 ± 0.27 nM) and a modest decrease in susceptibility to RPV (6.2 ± 1.6 nM). The Y181I mutant was also sensitive to DOR (0.63 ± 0.28 nM), but this mutant showed a reduced susceptibility to RPV (8.8 ± 0.12 nM). The double mutant K101P/V179I showed an increase in susceptibility to DOR (1.5 ± 0.4 nM); however, this mutant had a significant loss in susceptibility to RPV (93.5 ± 12.1 nM).
Antiviral activities of RPV and DOR against HIV-1 that contains mutations selected by RPV during cART and in cell culture. The IC50 values of RPV and DOR against viruses that contain WT RT and mutations selected by RPV in cell culture (E40K, D67E, K101E, and V111A) and in infected individuals during cART (K101E, E138K, M184I, M184V, K101E/M184I, K101E/M184V, E138K/M184I, and E138K/M184V) were measured using a single-round infection assay. Error bars represent the standard deviations of independent experiments (n = 4). The IC50 values of the graph have a maximum value of 100 nM and to better illustrate the lower IC50 values, the Y-axis was broken between 5 and 25 nM; there are equivalent breaks in the appropriate bar(s). The RPV susceptibility data were also used as a control in experiments testing the efficacy of a series of RPV analogs (21).
Antiviral Activities of RPV and DOR against HIV-1 that contains additional mutations selected by RPV in vivo. The IC50 values of RPV and DOR against viruses that contain WT RT and mutations selected by RPV in HIV-infected individuals were measured using a single round infection assay. Error bars represent the standard deviations of independent experiments (n = 4). The IC50 values of the graph have a maximum value of 100 nM and to better illustrate the lower IC50 values, the Y-axis was broken between 10 and 25 nM and in the appropriate bar. The RPV susceptibility data were also used as a control in experiments testing the efficacy of a series of RPV analogs (21).
Binding of RPV and DOR in the NNRTI Binding Pocket
Structural studies can be used to understand the binding of inhibitors to their cognate targets; HIV-1 RT has been crystallized with RPV and, more recently, with DOR.[16,17] RPV has a pyrimidine core with 2 aryl groups linked by amines–a benzonitrile group and dimethylphenyl moiety with a cyanovinyl, whereas DOR comprises a trifluoromethyl-pyridone core with a methyl-triazolone and a chlorophenol with a cyano group. The crystal structure of RPV in the NNRTI binding pocket showed that the chlorophenol moiety of RPV has aromatic stacking interactions with Y181 and the appended cyanovinyl extends into a hydrophobic tunnel located in the upper portion of the NNRTI binding pocket, formed by the sidechains of Y188, F227, W229, and L234. The amine linker nitrogens interact with the main chain carbonyl oxygens of K101 from the p66 subunit and E138 of the p51 subunit through hydrogen bonding. Residues L100, K103, and V179, which are located near the center of the NNRTI binding pocket interact with the central pyrimidine ring of RPV. The benzonitrile moiety makes contacts with Y318, P225, and P236. The interactions of DOR and the NNRTI binding pocket of RT in the crystal structure showed that the chlorophenol moiety stacks with Y188, and the DOR cyano moiety makes contacts that are similar to those made by RPV within the hydrophobic tunnel. The pyridone core of DOR interacts with V106 and L100, and the methyl triazolone stacks with P236 and interacts with the main chain atoms of K103.
Superimposing the structures of RT with DOR and RPV bound reveals significant differences in their binding (Supp. Fig. 2; Fig. 5) and the residues they contact in the binding pocket, which suggests how the 2 compounds differ in their susceptibility to NNRTI-resistant mutants. The superimposition shows that DOR resides ~1–2 Å deeper in the NNRTI binding site compared with RPV (Supp. Fig. 2). In the DOR structure (Fig. 5, right panel B), Y188 is ~4 Å closer to the middle and rim of the binding pocket to accommodate the chlorophenol moiety of DOR, whereas Y181 is ~8 Å closer to the rim of the binding pocket compared with the structure with RPV bound (Fig. 5, left panel A). Residue E138 moves ~4.5 Å away from the rim toward the back of the NNRTI binding pocket in the DOR structure as compared with the RPV structure, which prevents DOR from interacting with K101. In addition, the residues in the upper portion of the NNRTI binding pocket, L234, P225, and F227 are shifted by 1.5 Å in the structure with DOR bound compared with RPV RT structure. The substitution of the benzonitrile moiety of RPV with the triazolone of DOR causes a 2–3 Å movement of this substituent towards P236. The differences in the positions of these residues around the NNRTI binding pocket between the 2 structures matches the residues where mutations are likely to be selected by the 2 compounds. It appears, based both on the crystal structures and the behavior of the mutants in our antiviral assays, that residues in the upper portion of the binding pocket interact more extensively with DOR, and that the residues around the rim of the binding pocket interact more with RPV. RPV is known to change its binding in response to changes in the NNRTI binding pocket that are associated with the presence of drug resistance mutations. We do not yet have access to crystal structures of DOR bound to mutant forms of RT. It is possible that DOR is flexible to some extent; however, it is more broadly susceptible to drug resistance mutations than RPV.
Relative positions of RPV and DOR in the NNRTI binding pocket, showing contacts with specific residues. The crystal structures of RPV (left image, A) and DOR (right image, B) are displayed for comparison to illustrate the different contacts made between the inhibitor and the NNRTI binding pocket for each. In the RPV structure, the phenyl moiety of RPV stacks with Y181 (red circle), whereas E138 interacts through an electrostatic interaction with K101 (brown circle) and the benzonitrile moiety stacks with Y318 (orange circle). In the DOR structure, the chlorophenyl moiety of DOR alternatively stacks with Y188 (blue circle), whereas the triazolone functionality of DOR interacts with P236 (green circle), and residues E138 and Y181 (purple circle) move toward the rim and back of the NNRTI binding pocket, respectively. In both the RPV and DOR structures, the cyanovinyl and cyano functionalities, respectively, modified on the phenyl moiety extend in the hydrophobic pocket formed by the side chains Y188, F227, W229, and L234. In the figure, only E138K is from the RT p51 subunit in the RPV (black) and DOR (magenta) images. The remaining residues are from the RT p66 subunit in the RPV (dark green) and DOR (orange) structures.
J Acquir Immune Defic Syndr. 2016;72(5):485-491. © 2016 Lippincott Williams & Wilkins