Since the first agent, zidovudine, was licensed for the treatment of HIV-1 infection in 1987, a total of 25 drugs have been approved for the treatment of HIV (13
). Since 1996, the importance of anti-HIV drug combination regimens has become widely accepted, and in 2008, the Department of Health and Human Services guidelines (46
) recommend the inclusion of at least two, and preferably three, fully active antiretroviral agents when constructing drug regimens. Efavirenz-based therapy or boosted PI-based therapies are currently recommended as first-line regimens for the treatment of HIV infection. Although these recommendations provide effective treatment options for the majority of patients, treatment durability is still limited by drug-related side effects, inadequate patient adherence, and the development of drug resistance.
NNRTI-based regimens have been associated with a low genetic barrier to resistance, rash, hypersensitivity reactions, and central nervous system side effects. Etravirine, a novel NNRTI with activity against a broader panel of NNRTI mutant viruses, has recently been approved for use in treatment-experienced patients who have evidence of infection with HIV-1 strains that are resistant to an NNRTI and other antiretroviral agents (Intelence prescribing information; Tibotec Therapeutics, Raritan, NJ). However, rash and nausea are commonly reported adverse events in etravirine-treated subjects. There is, therefore, still a need for first-line antiviral agents that will facilitate patient adherence and allow durable suppression of viral replication.
In our laboratories, we have recently identified a novel inhibitor of HIV (11
). In this paper, we confirm that lersivirine belongs to the NNRTI class: in vitro
, it inhibits the RT enzyme by a mixed noncompetitive kinetic mechanism and binds the protein with a 1:1 stoichiometry in the NNRTI binding pocket. X-ray crystallography suggests a novel binding mode of lersivirine into the “NNRTI pocket,” which in turn translates to activity against key class resistance mutants.
A survey of more than 5,000 patient samples of NNRTI-treated individuals revealed that the most common mutations were K103N (29%), Y181C (14%), and G190A (17%) (6
). In agreement with this distribution, the most commonly occurring NNRTI mutations found in our panels of 353 isolates tested were K103N > Y181C/I > G190A/S. In this panel, lersivirine was active against all clinically derived viruses that had no NNRTI mutations, with many having phenotypic resistance to antiviral drugs of the NRTI and PI classes (data not shown).
Furthermore, lersivirine also retained inhibitory activity against a range of viruses carrying clinically relevant NNRTI mutations. Lersivirine inhibited 62% of isolates containing a single NNRTI mutation (in vitro resistance was defined using the BCO of 3.1) and was active against the majority of viruses carrying the single mutations K103N, Y181C, and G190A, which occur in the majority of patients failing NNRTI therapy (Table ). Although clinical studies have yet to confirm this finding in patients, these initial results suggest that lersivirine has the potential to be used in patients that failed a drug regimen containing efavirenz, nevirapine, or delavirdine because of mutations arising at location K103, Y181, or G190.
In our subtype study, one clade C virus (isolated in Puerto Rico) demonstrated a 6.3-fold IC50
to the reference virus strain. Population sequence analysis did not reveal the presence of the V106M mutation, which is often associated with resistance to efavirenz and NVP in clade C viruses, or of any other NNRTI resistance-associated mutations (21
). However, a number of amino acid changes were present in the RT gene from this patient virus, which may result in differences in susceptibility but not necessarily resistance. These include amino acids at positions 35, 135, 158, and 245 (9
; J. Whitcomb, personal communication). Slight decreases in susceptibility were also observed when this isolate was tested against efavirenz and nevirapine (1.9- and 2.2-fold changes from those for the reference virus, respectively).
Sequential passage experiments with wt virus in the presence of progressively increasing concentrations of drug in vitro
can identify the accumulation of mutations that confer resistance. Lersivirine dose escalation studies identified two pathways to resistance; in both cases V108I and F227L were selected, with resistance increased further as a result of the emergence of E138K or M230M/I. The V108I mutation was also selected in fixed-dose studies using the wt and viruses with preexisting NNRTI-resistant mutations. Acquisition of a V108I mutation appears to be the preferred molecular pathway to lersivirine resistance and differs from that of efavirenz, etravirine, and rilpivirine. Two different pathways to resistance were reported following dose escalation selection experiments with WT virus for etravirine: V179F plus Y181C plus M230L and Y181C plus G190E (49
). In addition, the Y181C mutation was selected in etravirine fixed-dose studies, suggesting the importance of this mutation in the development of resistance to etravirine (49
Several pathways were identified following escalating dose concentrations of rilpivirine; combinations of the mutations L100I, K101E, Y181C, and F227V were frequently seen with the laboratory strain IIIB (4
). The preferred molecular pathway to lersivirine resistance would appear to differ from that of etravirine and rilpivirine. The most common NNRTI resistance-associated substitutions (seen in at least 10% of patients) that emerged in patients with virologic failure—who received etravirine in the DUET-1 and -2 studies—included V108I, V179F, V179I, and Y181C. These usually emerged in a background of other NNRTI resistance-associated substitutions (20
Interestingly, phenotypic data indicated that multiple mutations were required to establish substantial resistance to lersivirine. In both dose escalation and fixed-dose studies V108I was selected, suggesting this variant to be the preferred molecular pathway under lersivirine drug pressure. Phenotypically, the V108I mutant showed no cross-resistance to efavirenz or etravirine. Similarly, lersivirine-resistant viruses generated by dose escalation showed no cross-resistance to efavirenz and DLV. These results suggest that resistance was associated with, and specific for, the presence of lersivirine and may not lead to class-wide resistance.
The question arises as to what makes lersivirine resilient against drug resistance mutations. While it is difficult to unambiguously rationalize the effect of mutations in the NNRTI binding pocket with changes in IC50
, the three-dimensional structure that we describe allows us to propose a hypothesis. As shown in Fig. , lersivirine makes extensive contacts through the 3,5-dicyanophenoxy group with Trp229 at the top of the binding pocket. This residue not only is part of the NNRTI binding pocket but also belongs to the “primer grip” of HIV RT, and any mutation at this position has been shown to severely compromise the RNA- and DNA-dependent DNA polymerase activities of the enzyme (33
). Indeed, this immutable residue has become the focus of the development of novel NNRTIs (17
). In addition, upon the binding of many NNRTIs, the side chain of Tyr181, contained within the β4-β7-β8 sheet, have been reported to flip conformation from a “down” to an “up” position (38
). Upon the binding of lersivirine into the NNRTI binding pocket, the Tyr181 is in the less frequently observed “down” conformation. This tyrosine does not make the equivalent contacts with the disubstituted group that is observed with other NNRTIs, such as capravirine (37
). The resilience of lersivirine to the Y181C mutation can be explained by a reduction in the aromatic stacking interactions with these residues. A recently reported benzophenone NNRTI series also takes advantage of this alternative Y181 conformation (35
Finally, we also observed that specific polar interactions between RT and lersivirine are limited to the hydrogen bonds formed to the backbone amides of the protein. We found that lersivirine accepts a hydrogen bond from the Lys103 amide and donates a hydrogen bond to the Pro236 carbonyl. No specific interaction is made with the 103 side chain, and no significant change in compound binding to the mutant enzyme is observed. In short, lersivirine is an expression of a minimal NNRTI pharmacophore in which maximum advantage is taken of the regions of the binding sites that are constant while minimizing contacts with regions of the site that are known to mutate. This strategy differs from that adopted for higher-molecular-weight NNRTIs, such as etravirine, where mutations are compensated for by a larger contact area. The susceptibility of lersivirine to the V108I mutant (a 5-fold decrease in potency) can be rationalized as this residue makes extensive van der Waals contact with the dicyano-substituted phenol ring (4.0-Å contact distance to the ring). A sterically more demanding isoleucine at this position could perturb the binding mode observed.
Taking these data into consideration, lersivirine should be a potent agent active against key clinical mutant HIV-1 strains. Indeed, when the EC50
for lersivirine is compared to the steady-state pharmacokinetics of lersivirine in HIV-1-infected patients receiving the compound as monotherapy at 500 mg once a day (QD) or 750 mg QD, there is a 50- and 93-fold window, respectively, between the protein-adjusted wt geometric mean inhibition in vitro
and the average free drug levels achieved (Fig. ) (15
). Interestingly, at these doses, the average free concentrations of lersivirine would be expected to be well above the EC50
for most mutants studied.
FIG. 5. Comparison of the free steady-state average concentrations (Cavg) in the monotherapies with 500 mg QD (A) and 750 mg QD (B) in HIV patients and activity of lersivirine (EC50 potency) against wt and mutant NL4-3 viruses (Fig. ). Target-free (more ...)
To support the use of lersivirine as part of a multidrug regimen, the antiviral activity of lersivirine was evaluated in combination with representatives from each of the licensed antiretroviral classes in vitro
. Preliminary data indicate additive antiviral interactions with all compounds in combination with lersivirine (with the exception of the NRTIs, which frequently demonstrated synergistic interactions). Synergy has previously been reported between NRTIs and NNRTIs which target the same viral enzyme. Zidovudine and efavirenz were shown to be synergistic in cell culture, and studies of etravirine combined with zidovudine suggested synergy, though additive effects were reported when etravirine was combined with other NRTI drugs (abacavir, didanosine, lamivudine, stavudine, zalcitabine, zidovudine, and tenofovir) and representatives of the PIs and the currently marketed NNRTIs (2
). The synergistic interaction between NRTIs and NNRTIs has been associated with the ability of the NNRTI to inhibit ATP-mediated removal of the NRTI and so prolong its MOA (7
). The synergistic behavior of NRTIs and NNRTIs in combination in vitro
may contribute to their effectiveness in vivo
There was no evidence of antagonistic interactions with any of the compounds investigated, and there was no evidence of synergistic cytotoxicity at the highest concentrations tested.
In conclusion, lersivirine represents an attractive novel NNRTI with potency against an interesting panel of key clinical NNRTI mutant HIV-1 viruses. Ongoing clinical trials (15
) are investigating its potential for treatment of HIV-1-infected patients in the context of HAART.