Rilpivirine and Doravirine Have Complementary Efficacies Against NNRTI-Resistant HIV-1 Mutants

Abstract

Background: Rilpivirine (RPV) is the latest non-nucleoside reverse transcriptase inhibitor (NNRTI) to be approved by Food and Drug Administration to combat HIV-1 infections. NNRTIs inhibit the chemical step in viral DNA synthesis by binding to an allosteric site located about 10 Å from the polymerase active site of reverse transcriptase (RT). Although NNRTIs potently inhibit the replication of wild-type HIV-1, the binding site is not conserved, and mutations arise in the binding pocket. Doravirine (DOR) is a new NNRTI in phase III clinical trials.

Methods: Using a single round HIV-1 infection assay, we tested RPV and DOR against a broad panel of NNRTI-resistant mutants to determine their respective activities. We also used molecular modeling to determine if the susceptibility profile of each compound was related to how they bind RT.

Results: Several mutants displayed decreased susceptibility to DOR. However, with the exception of E138K, our data suggest that the mutations that reduce the potency of DOR and RPV are non-overlapping. Thus, these 2 NNRTIs have the potential to be used together in combination therapy. We also show that the location at which DOR and RPV bind with the NNRTI binding pocket of RT correlates with the differences in their respective susceptibility to the panel of NNRTI-resistance mutations.

Conclusions: This shows that (1) DOR is susceptible to a number of well-known NNRTI resistance mutations and (2) an understanding of the mutational susceptibilities and binding interactions of NNRTIs with RT could be used to develop pairs of compounds with non-overlapping mutational susceptibilities.

Figure 1. Antiviral activity of DOR and RPV against HIV-1 vectors that carry well-known NNRTI resistance mutations

(A) The IC₅₀ values of DOR against WT HIV-1 and several NNRTI-resistant mutants, represented in different colors, were measured using a single round infection assay. (B) The IC₅₀ values of RPV and DOR against WT HIV-1 and several other well-characterized NNRTI resistant mutants, represented in different colors, were measured using a single round infection assay. The IC₅₀ value of DOR against the M230L, K103N/P225H, and V106A/G190A/F227L resistant mutants were >100 nM. Error bars represent the standard deviations of independent experiments, (n=4). The RPV susceptibility data were also used as a control in experiments testing the efficacy of a series of RPV analogs (21).

Figure 2. 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).

Figure 3. 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).

Figure 4. 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).

Figure 5. 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), while 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), while 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.