Rilpivirine analogs potently inhibit drug-resistant HIV-1 mutants
© Smith et al. 2016
Received: 9 December 2015
Accepted: 5 February 2016
Published: 16 February 2016
Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a class of antiretroviral compounds that bind in an allosteric binding pocket in HIV-1 RT, located about 10 Å from the polymerase active site. Binding of an NNRTI causes structural changes that perturb the alignment of the primer terminus and polymerase active site, preventing viral DNA synthesis. Rilpivirine (RPV) is the most recent NNRTI approved by the FDA, but like all other HIV-1 drugs, suboptimal treatment can lead to the development of resistance. To generate better compounds that could be added to the current HIV-1 drug armamentarium, we have developed several RPV analogs to combat viral variants that are resistant to the available NNRTIs.
Using a single-round infection assay, we identified several RPV analogs that potently inhibited a broad panel of NNRTI resistant mutants. Additionally, we determined that several resistant mutants selected by either RPV or Doravirine (DOR) caused only a small increase in susceptibility to the most promising RPV analogs.
The antiviral data suggested that there are RPV analogs that could be candidates for further development as NNRTIs, and one of the most promising compounds was modeled in the NNRTI binding pocket. This model can be used to explain why this compound is broadly effective against the panel of NNRTI resistance mutants.
KeywordsHIV-1 Nonnucleoside reverse transcriptase inhibitors Rilpivirine Antiviral activity Resistance Analogs Susceptibility Binding pocket Doravirine
HIV-1 reverse transcriptase (RT) is a target for many drugs used in highly active antiretroviral therapy (HAART) to treat HIV-1 infections . RT has two enzymatic activities: (1) a DNA polymerase that can copy either an RNA or a DNA template and (2) an RNase H that degrades RNA if and only if the RNA is part of a RNA: DNA hybrid. Although there are, as yet, no drugs that target the RNase H of HIV-1 RT, there are two classes of drugs that target the DNA polymerase. The first class consists of inhibitors that are analogs of normal nucleosides used to synthesize DNA (nucleoside reverse transcriptase inhibitors, NRTIs). All of the FDA-approved NRTIs lack the 3′ –OH present on the deoxyribose of normal nucleosides. When an NRTI is incorporated into the growing viral DNA strand, it acts as a chain terminator. The second class consists of inhibitors that bind in a small hydrophobic pocket ~10 Å from the polymerase active site (non-nucleoside reverse transcriptase inhibitors, NNRTIs) [2–4]. The NNRTI binding pocket is closed in the absence of a bound NNRTI . The NNRTI binding pocket is formed by the following residues: L100, K101, K103, V106, V179, Y181, Y188, G190, F227, W229, L234, P236, and Y318. The binding pocket lies underneath the bound double-stranded nucleic acid substrate. Binding of an NNRTI distorts RT, which affects the alignment of the primer terminus and the polymerase active site, blocking the chemical step of viral DNA synthesis [6–8]. There currently are sixteen drugs that target RT that have been approved by the FDA for the treatment of HIV-1 infections; eleven are NRTIs and five are NNRTIs. The five approved NNRTI drugs are: nevirapine (NVP, Viramune), delavirdine (DLV, Rescriptor), efavirenz (EFV, Sustiva), etravirine (ETR, Intelence), and rilpivirine (RPV, Endurant,). While NNRTIs effectively block the replication of WT HIV-1, NNRTI-resistant mutants can emerge during treatment, many of which cause cross-resistance among the approved NNRTIs.
There are two primary factors that contribute to the emergence of resistance to NNRTIs: (1) HIV-1 RT can tolerate a wide range of sequences in and around the NNRTI binding pocket and (2) there is extensive HIV-1 genetic variation [9, 10]. Although most HIV-infected individuals are initially infected with a single virion, HIV-1 variants arise rapidly due to high viral loads in HIV-1 infected patients, which leads to the infection of large numbers of cells, the rapid turnover of these infected cells, and to errors made during HIV-1 replication [10–12]. Ultimately, error prone replication creates the mutations that enable HIV-1 to develop resistance against antiretroviral drugs.
A number of drug resistant mutants were selected in HIV-infected individuals by the first generation NNRTIs (NVP, EFV, and DLV). Resistance mutations are commonly seen in the residues that surround the NNRTI binding pocket, including: L100I, K103N, V106A, Y181C, Y188L, and H221Y. These mutations alter the geometry of the NNRTI binding pocket in ways that interfere with the binding of NNRTIs. The first generation inhibitors were rigid and bulky compounds that were particularly vulnerable to the effects of resistance mutations. The second generation NNRTIs, ETR and RPV, were designed to be less bulky and more flexible, and are better able to adapt to the changes in the NNRTI binding pocket caused by resistance mutations. This allows these newer NNRTIs to effectively inhibit both WT HIV-1 and a number of drug resistant variants [13, 14]. However, when these drugs were tested in clinical trials, patients who were on an ETR containing regimen were found to have a combination of mutations including: V90I, A98G, L100I, K101E/P, V106I, V179D/F, Y181C/I/V, and G190A/S . A variety of mutations including K101E/P, E138A/G/K/Q/R, Y181C/I/V, and M230L have been shown to be associated with a decrease in susceptibility to RPV and have been detected in clinical samples from HIV-1-infected individuals treated with RPV . Individuals who failed in a clinical trial involving therapy with RPV and NRTIs had mutations at positions E138K and M184I/V; the M184V mutation was likely selected by FTC, an NRTI often included in HAART regimens [17, 18].
Because development of resistance can occur with all of the available NNRTIs, there are ongoing efforts to develop NNRTIs that extend and improve the effectiveness of the previously identified compounds. For example, doravirine (DOR) is a new NNRTI in late stage clinical trials . Although it selects for resistance, the mutations that reduce the potency of DOR do not, for the most part, overlap with mutations that reduce the potency of RPV . As an alternative approach, we are testing whether RPV analogs have better resistance profiles than the parent compound.
Several of our RPV analogs are broadly effective against HIV-1 mutants. One compound (11) was particularly effective in inhibiting the replication of mutants that have reduced susceptibility to RPV and DOR, suggesting that it has the potential to become a clinically useful NNRTI.
Results and discussion
Susceptibility of mutant HIV-1 to RPV analogs
We previously determined the ability of several of our RPV analogs to inhibit infection of WT HIV-1 or mutants that contain several well-known NNRTI-resistant mutations (L100I, K103N, E138K, Y181C, Y188L, H221Y, and K103N/Y181C) using a single-round infection luciferase assay . As shown in Figs. 3 and 4 of that aforementioned study, RPV and several analogs (6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 21, and 27) had potent antiviral activities against both WT HIV-1 and several of the resistant mutants L100I, K103N, Y181C, and H221Y (all IC50s < 7 nM). The E138K mutant caused only a small drop in the IC50s for most of the compounds (<5 nM); however there was a greater loss in susceptibility to 12 (15 nM ± 1.2). The resistant mutant Y188L showed modest drop in susceptibility to 8, 9, 11, 12, 14, 15, 16, and 17 (IC50s ranged from 12 to 64 nM), whereas compounds 6, 7, 13, 21, and 27 showed a modest loss of potency against this resistant mutant (all <10 nM). The NNRTI resistant double mutant K103N/Y181C caused a substantial decrease in susceptibility to RPV analogs 6, 8, 9, 13, 21, and 27 (ranged from 25 to 100 nM), whereas this mutant caused a minor loss in susceptibility to RPV, 7, 11, and 13 (<5 nM).
Susceptibility of mutants selected by DOR to inhibition by RPV analogs
Susceptibility of viruses carrying RPV-resistance mutations to the RPV analogs
Modeling the binding of 11 to HIV-1 RT
RPV is the most recent NNRTI to be FDA-approved for the treatment of HIV-1 infection. It was designed and developed to effectively inhibit the replication of a number of common NNRTI-resistant mutants. RPV is comprised of three linked rings, and it is smaller than the largest compounds that can bind in the NNRTI binding pocket. As a consequence, it has the ability, when bound to mutant RTs, to undergo conformational changes and shifts in its binding position that allow it to bind tightly to both the WT and mutant forms of RT, altering its conformation to adapt to the changes in the geometry of the binding pocket. However, recent data suggest that RPV is susceptible to some NNRTI binding pocket mutations [16–18]. We have made additional RPV analogs to determine whether structural changes would allow the new derivatives to be more effective against mutants that reduce the efficacy of RPV.
To evaluate the potential of our RPV analogs relative to RPV, the related drug ETR, and DOR, we examined antiviral efficacies of the compounds against a broad panel of NNRTI-resistant HIV-1 mutants. Most of the RPV analogs exhibited excellent antiviral activities against mutants that were selected by first generation NNRTIs and the new compound DOR, which is in late stage clinical trials . In particular, most of the RPV analogs displayed strong antiviral activities against mutants that contain E138K. More importantly, a double mutant, K101P/Y179I, which showed a significant drop in susceptibility to RPV, remained susceptible to analog 11, demonstrating that it has an antiviral profile that is broader that any of the NNRTIs we have tested thus far.
Different mutation can arise at position Y181, including Y181I and Y181C. Y181C is susceptible to RPV and RPV analogs as previously described . However, Y181I caused a substantial decrease in susceptibility to the RPV analogs, except for 11 and 13 (2.4 nM ± .6 and 1.2 nM ± .06, respectfully); a minor drop in susceptibility to RPV (8.8 nM ± .12) was observed. The isoleucine side chain at position 181 could cause a steric clash with RPV and most of the RPV analogs that would prevent an interaction with Y183 and the cyanovinyl modification. This interaction is thought to compensate for the disruption of the π-π stacking interaction between the aromatic side chain of Y181 and the phenyl moiety of RPV (and most of the RPV analogs). The interaction with Y183 can still occur with the Y181C mutant .
11 has a structure that is distinct from the other RPV analogs. The central 2,6-purine ring system and the aryl amines of 11 are in a similar conformation to the central pyrimidine ring of the parent compound RPV, as opposed to 12, 13, 14, 15, 16, 17, and 21, which are in a “flipped” conformation. In this arrangement, the cyanovinyl functionality can extend deeper into the hydrophobic tunnel and the benzonitrile moiety can move more towards the back of the NNRTI binding pocket. In addition to the deeper binding of the aryl moieties of 11, relative to RPV, the overall structure and size of 11 could allow it (like RPV) to use its torsional flexibility in the NNRTI binding pocket in response to mutations in the NNRTI binding pocket [14, 28].
The wide range of NNRTI-resistant mutants that have been identified shows how daunting the challenges are for designing new NNRTIs for the treatment of HIV-1 infection. More and better compounds should be developed to achieve the goal of suppressing the emerging resistant HIV-1 mutants. One possible approach is to identify combinations of two NNRTIs that have non-overlapping resistance profiles and then use them in combination (unpublished observations), rather than trying to prepare a single compound that is effective against the growing array of NNRTI-resistant mutants . Preventing the emergence HIV-1 resistance is a much better and safer treatment strategy than attempting to deal with resistant mutants after they have been selected. However, that strategy does not mean that we should abandon the search for more effective compounds. NNRTIs offer better toxicity profiles than NRTIs and protease inhibitors (PIs) [30, 31]. 11 and 27 represent a promising step in what should be an ongoing process for the development of future NNRTIs. It also appears, based on what is now known, that DOR, or perhaps some improved DOR derivative, could be useful if it was given in combination with these RPV analogs.
The human osteosarcoma cell line, HOS, was obtained from Dr. Richard Schwartz (Michigan State University, East Lansing, MI) and grown in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 5 % (v/v) fetal bovine serum, 5 % newborn calf serum, and penicillin (50 units/ml) plus streptomycin (50 µg/ml; Quality Biological, Gaithersburg, MD). Virion production and single-round infectivity assays were used to determine antiviral activity (IC50 values) of the compounds as previously described .
Selection of NNRTI mutations in HIV-1
HuT-CCR5 cells were maintained in RPMI 1640 medium (Life Technologies), 10 % fetal bovine serum (Atlanta Biologicals), 100 U/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies), 0.292 mg/ml l-glutamine (Life Technologies), 0.5 μg/ml puromycin (EMD Millipore), and 100 μg/ml G418 (Life Technologies). HuT-CCR5 cells were infected with HIV-1LAI for 2 h at a multiplicity of infection of 0.1–0.01. Cells were cultured in the presence or absence of RPV, analog 7, or analog 11. Viral RNA from the supernatant was isolated and used as template in RT-PCR assays with primers spanning the RT coding region. PCR product was sequenced to identify any RT mutations.
pNLNgoMIVR-ΔEnv.LUC has been described previously . The RT open reading frame was removed from pNLNgoMIVR-ΔEnv.LUC (digestion with SpeI and SalI) and inserted between the SpeI and SalI sites of pBluescript II KS+. Using that construct as the wild-type template, we prepared the following HIV-1 RT mutants using the QuikChange II XL (Stratagene, La Jolla, CA) site-directed mutagenesis protocol kit: L100I, K103N, Y181C, Y188L, H221Y, K103N/Y181C, G190A, G190S, M230L, P236L, L100I/K103N, K103N/P225H, V106A/G190A/F227L, V106A, L234I, V106A/F227L, V106A/L234I, V106A/F227L/L234I, E40K, D67E, K101E, V111A, E138K, M184I, M184V, K101E/M184I, K101E/M184V, E138K/M184I, E138K/M184V, K101P, Y181I, and K101P/V179I. The following sense oligonucleotides were used with matching cognate antisense oligonucleotides (not shown) (Integrated DNA Technologies, Coralville, IA) in the mutagenesis: L100I, 5′-CATCCCGCAGGGATAAAAAAGAAAAAATCA-3′; L100I 2-103, 5′-ATACCACATCCCGCAGGGATTAAAAAGAATAAATCAGTA-3′; K103N, 5′-GCAGGGTTAAAAAAGAATAAATCAGTAACAGTA-3′; Y181C, 5′-CCAGACATAGTTATCTGTCAATACATGGATGAT-3′; Y188L, 5′-TACATGGATGATTTGCTAGTAGGATCTGACTTA-3′; H221Y, 5′-ACACCAGACAAAAAATATCAGAAAGAACCTCCA-3′; G190A, 5′-ATGGATGATTTGTATGTAGCATCTGACTTAGAAATAGGG-3′; G190S, 5′-ATGGATGATTTGTATGTAAGTTCTGACTTAGAAATAGGG-3′; P225H, 5′-AAAAAACATCAGAAAGAACATCCATTCCTTTGGATGGGT-3′; F227, 5′-CATCAGAAAGAACCTCCATTACTTTGGATGGGTTATGAA-3′; M230L, 5′-GAACCTCCATTCCTTTGGCTGGGTTATGAACTCCATCCT-3′; P236L, 5′-ATGGGTTATGAACTGCATCTCGATAAATGGACAGTACAG-3′; V106A, 5′-AAAAAGAAAAAATCAGCAACAGTACTGGATGTG-3′; L234I, 5′-TTCCTTTGGATGGGTTATGAAATCCATCCTGATAAATGGACAGTA-3′; E40K, 5′-GAAATTTGTACAAAAATGGAAAAGGAAGGG-3′; D67E, 5′-GCCATAAAGAAAAAAGAAAGTACTAAATGGAGA-3′; K101E, 5′-CATCCCGCAGGGTTAGAAAAGAAAAAATCAGTAACA-3′; V111A, 5′-GTAACAGTACTGGATGTAGGTGATGCATATTTTTCA-3′; E138K, 5′-CCTAGTATAAACAATAAGACACCAGGGATTAGA-3′; M184I, 5′-GTTATCTATCAATACATAGATGATTTGTATGTA-3′; M184V, 5′-GTTATCTATCAATACGTTGATGATTTGTATGTA-3′; K101P, 5′-CCACATCCCGCAGGGTTACCAAAGAAAAAATCAGTAACA-3′; Y181I, 5′-AATCCAGACATAGTTATCATTCAATACATGGATGATTTG-3′; K101P/V179I, 5′-AAACAAAATCCAGACATAATCATCTATCAATACATGGAT-3′. The double mutants K103N/Y181C, K103N/P225H, and L100I/K103N were made using the previously generated K103N mutant and the appropriate oligonucleotides to add the second mutation, Y181C, P225, and L100I respectively. The double mutants V106A/F227L and V106A/L234I were prepared using the previously generated V106A mutant and the appropriate oligonucleotides to add the second mutation, F227L and L234I, respectively. The double mutants K101E/M184I and K101E/M184V were constructed using the previously generated K101E mutant and the appropriate oligonucleotides to add the second mutation, M184I and M184V, respectively. The double mutants E138K/M184I and E138K/M184V were constructed using the previously generated E138K mutant and the appropriate oligonucleotides for the second mutation, M184I and M184V, respectively. The triple mutant V106A/F227L/L234I was made using the previously generated V106A/F227L double mutant and the appropriate oligonucleotides for the third mutation, L234I. The triple mutant V106A/G190A/F227L was constructed in a series of steps using the previously generated V106A mutant and the appropriate oligonucleotides for the second mutation, G190A, to generate the double mutant V106A/G190A, and then the triple mutant was made using double mutant V106A/G190A and oligonucleotides for the third mutation, F227L. The DNA sequence of each construct was verified by DNA sequence determination. The sequences encoding the mutant RTs were then subcloned into pNLNgoMIVR-ΔEnv.LUC (between the KpnI and SalI sites) to produce the mutant HIV-1 constructs. These DNA sequences were also checked independently by DNA sequence determination.
All computer modeling was performed using MOE2014.09 (Chemical Computing Group, Montreal, Quebec, Canada). The previously reported crystal structure of WT HIV-1 RT/RPV complex (PDB ID: 2ZD1; ) was used in the docking stimulation to model the RPV analogs in the NNRTI binding pocket. The docking simulation used a rigid receptor protocol and refinement to predict the pose of the RPV analogs in the NNRTI binding pocket. We also used an induced fit docking protocol and refinement that permits the sidechains of the NNRTI binding pocket to move to determine whether this would affect predicted binding of the RPV analogs in the NNRTI binding pocket. No new poses were detected.
SS performed the infectivity assays and conducted the molecular modeling. SS and AA made the vector constructs and DNA preparations. KM conducted NNRTI selection experiments. GP, CT, GR, DM, and JS contributed to synthesis of compounds. SS, ZA, and SH designed the experiments. SS and SH drafted the manuscript. All authors read and approved the final manuscript.
The authors would like to thank Teresa Burdette for help in preparing the manuscript and Joseph Meyer for help preparing the figures. This research was supported by the Intramural Research Programs of the National Cancer Institute, the National Human Genome Research Institute, the National Center for Advancing Translational Sciences, the Intramural AIDS Targeted Antiviral Program (IATAP), and NIH Grants R01 AI080290 (ZA) and T32 AI065380 (KM).
The authors declare that they have no competing interests.
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