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Antiviral Drug Resistance and the Need for Development of New HIV-1 Reverse 1
Transcriptase Inhibitors. 2
3
Eugene L. Asahchop1, 2, 3, Mark A. Wainberg1*, Richard D. Sloan1 and Cécile L. 4
Tremblay2, 3. 5
6
1. McGill University AIDS Centre, Lady Davis Institute, Jewish General Hospital, 7
Montreal, Quebec, Canada. 8
2. Centre Hospitalier de I 'Université de Montréal, Montréal, Québec, Canada. 9
3. Département de Microbiologie et d’Immunologie, Université de Montréal, Montréal, 10
Québec, Canada. 11
12
*Corresponding Author: 13 Dr. Mark A. Wainberg 14 McGill University AIDS Centre 15 Jewish General Hospital 16 3755, Cote-Ste-Catherine-Road 17 Montreal, Quebec 18 H3T 1E2 19 CANADA 20 Telephone Number: (514) 340-8260 21 FAX Number: (514) 340-7537 22 E-mail: mark.wainberg@mcgill.ca 23 24
25
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28
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00591-12 AAC Accepts, published online ahead of print on 25 June 2012
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Abstract 31
Highly active antiretroviral therapy (HAART) consists of a combination of drugs to achieve 32
maximal virological response and reduce the potential for the emergence of antiviral 33
resistance. Despite being the first effective antivirals described to be effective against HIV, 34
reverse transcriptase inhibitors remain the cornerstone of HAART. There are two broad 35
classes of reverse transcriptase inhibitor, the nucleoside reverse transcriptase inhibitors 36
(NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). 37
Since the first such compounds were developed, viral resistance to them has inevitably been 38
described; this necessitates the continuous development of novel compounds within each 39
class. In this review we consider the NRTIs and NNRTIs currently in both pre-clinical and 40
clinical development or approved for second line therapy, and describe the patterns of 41
resistance associated with their use, as well as the underlying mechanisms that have been 42
described. Due to reasons of both affordability and availability, some reverse transcriptase 43
inhibitors with low genetic barrier are more commonly used in resource limited settings. 44
Their use results to the emergence of specific patterns of antiviral resistance and so may 45
require specific actions to preserve therapeutic options for patients in such settings. 46
47
48
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Introduction 49
The standard treatment for patients infected with human immunodeficiency virus (HIV), 50
referred to as highly active antiretroviral therapy (HAART), consists of three or more HIV 51
drugs, most commonly two nucleoside reverse transcriptase inhibitors (NRTIs) in 52
combination with either a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease 53
inhibitor (PI) or, more recently, an integrase inhibitor (INI) (65). The goal of HAART is to 54
optimally suppress HIV replication during long-term therapy and to maintain immune 55
function (92). Rational drug selection is essential to maximize potency, minimize side effects 56
and cross resistance, preserve future treatment options, and increase overall duration of viral 57
suppression [reviewed in (23)]. Although numerous antiretroviral combinations may provide 58
potent suppression of viral replication, therapeutic choices necessitate careful consideration 59
of the potential impact of viral resistance on subsequent treatment options. 60
61
Advances in antiretroviral therapy have improved HIV management and the control of the 62
spread of regional epidemics (64). However, resistance to antiretroviral drugs is largely 63
unavoidable, due to the error-prone nature of HIV reverse transcriptase (RT), and its lack of a 64
proofreading function (77). In addition, the sheer number of replication cycles occurring in an 65
infected individual and the high rate of RT-mediated recombination events, facilitate the 66
selection of drug resistant mutant strains of HIV (13, 28). Furthermore, certain tissue 67
compartments seem able to select for resistance mutations due to the presence of low drug 68
concentrations (33). These mutations are located on the genes that encode antiretroviral 69
targets such as RT, resulting in the production of RT that is different than its wild-type (wt) 70
counterpart in both structure and function. Although this protein is still able to play its role in 71
HIV replication, it is not inhibited as effectively as wt protein by the ARV drugs. 72
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The number of mutations required for resistance to occur varies from drug to drug. Many 73
factors determine the relative rate of resistance selection with different drugs and drug 74
combinations, and this is reflected in the `genetic barrier' to resistance which refers to the 75
number of mutations that must occur within a given target in order for resistance to be present 76
against a particular drug. Interactions between mutations, the effects of individual resistance 77
mutations on viral replication capacity, and viral fitness all influence mutational pathways 78
and the overall impact of resistance mutations on viral phenotype. Many different 79
mechanisms through which HIV-1 escapes from drug pressure have been described; these 80
mechanisms differ from one drug class to another and can even differ between drugs of the 81
same class. 82
83
Reverse Transcriptase (RT) Inhibitors 84
Two classes of RT inhibitors exist; the nucleoside reverse transcriptase inhibitors (NRTIs) 85
and non-nucleoside reverse transcriptase inhibitors (NNRTIs). NRTIs incorporate into 86
nascent viral DNA, resulting in DNA chain termination and blocking further extension of 87
DNA. The NNRTIs stop HIV-1 replication by binding to the hydrophobic pocket within the 88
p66 sub unit of the RT enzyme thus preventing it from converting viral RNA into DNA (17, 89
74). NNRTIs are non-competitive inhibitors of HIV-1 RT and do not require activation. The 90
low fidelity of HIV-1 RT, high level of HIV-1 replication and the high rate of RT mediated 91
recombination collectively contribute to the emergence of resistance to RT inhibitors (10, 92
28). 93
94
Early NRTIs 95
HIV can become resistant to NRTIs via two distinct mechanisms. The first is discrimination, 96
whereby the mutated viral RT can selectively avoid incorporating NRTIs in favor of natural 97
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dNTPS; this mechanism is typified by such mutations as K65R, L74V, Q151M and M184V 98
(37). The second mechanism of resistance allows a mutated RT to enact the phosphorolytic 99
excision of NRTIs from the 3’ end of the viral DNA chain that extends from the primer, a 100
process referred to as ‘primer unblocking’. Examples of mutations involved in this process 101
are those selected by zidovudine (ZDV) and stavudine (d4T), that are termed thymidine 102
analogue mutations (TAMs), e.g. M41L, D67N, K70R, L210W, T215Y/F and K219Q/E (48, 103
68). TAMs confer resistance to all NRTIs except lamivudine (3TC) and emtricitabine (FTC). 104
Although, there is a degree of cross-resistance associated with TAMs, ultimate levels of 105
resistance depend on the specific NRTI and the number of TAM mutations found in the viral 106
RT (reviewed in (48)). 107
The two NRTI resistance mechanisms of discrimination and excision can also influence each 108
other. For example, the M184V/I mutation that is selected by 3TC and FTC is a 109
discrimination mutation but viruses that contain M184V/I are less likely to quickly develop 110
TAMs under selective pressure with such drugs as ZDV. Viruses containing M184V/I are 111
also more susceptible to ZDV and d4T than wild-type viruses (reviewed in(48)). 112
113
Antiretroviral therapy with abacavir (ABC) leads to the selection of such mutations as K65R, 114
L74V, Y115F and M184V (53) and the combination of L74V and M184V is commonly 115
observed. The K65R mutation is also selected by tenofovir (TFV) and reduces susceptibility 116
by 3 to 6-fold against this drug (8, 67). Usually, the selection of K65R precludes the 117
occurrence of TAMs while the presence of the latter mutations prevents the selection of 118
K65R due to the fact that viruses that contain both K65R and TAMs are not viable. The gold 119
standard in patients initiating therapy involves a combination of TFV/FTC or ABC/3TC 120
together with a NNRTI (reviewed in (75)). 121
122
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Early NNRTIs 123
Nevirapine (NVP) and Efavirenz (EFV) are FDA approved NNRTIs and have become the 124
cornerstone of therapy within both developed and under-developed countries. However, the 125
low genetic barrier to resistance of these earlier NNRTIs serves as a major limitation for 126
prolonged antiretroviral therapy and sequential use of inhibitors of this class (2, 7, 42). 127
Notably, a single amino acid substitution in the RT enzyme is often adequate to yield high-128
level clinically relevant resistance. Additionally, high-level cross-resistance among early 129
NNRTIs has been reported (7) and this can also have an impact by decreasing virologic 130
response in patients with transmitted resistance (44). The prevalence rate of transmitted 131
antiretroviral drug resistance in treatment-naïve patients with HIV-1 has been estimated to be 132
5-15% for resistance mutations to at least one antiretroviral class (6). In this US drug 133
resistance survey, the NNRTI class showed the highest prevalence at a rate of 6.9% compared 134
to NRTIs and PIs at 3.6% and 2.4% respectively. The increasing use of NNRTIs in clinical 135
practice and the fact that NNRTI resistance mutations do not severely impair viral replication 136
capacity but remain part of the dominant viral variant may explain the high prevalence of 137
NNRTI resistance [reviewed in (20)]. 138
139
The NNRTI binding pocket is located largely in the p66 subunit of RT and consists of the 140
following residues; 95, 100, 101, 103, 106, 108, 179, 181, 188, 190, 227, 229, 234, 236 and 141
318 (36, 74). Some residues from p51, such as 138, also contribute to the NNRTI binding. 142
NNRTI mutations confer resistance by disrupting the interactions between the inhibitor and 143
enzyme. This can occur through three mechanisms: 1. they can block the entry of inhibitor 144
into the NNRTI binding pocket (e.g K103N); 2. they can affect contacts between the inhibitor 145
and residues that line the NNRTI binding pocket (e.g Y181C), or 3. they can alter the 146
conformation or size of the NNRTI binding pocket so that it becomes less specific for the 147
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inhibitor (e.g Y188L) (20). Some resistance mutations can affect the binding of NNRTIs 148
through more than one mechanism. 149
150
Newer NRTIs 151
Elvucitabine 152
Elvucitabine (Table 1) is a novel NRTI currently in late phase II study and was developed by 153
Achillion Pharmaceuticals. In an in vitro selection study, only two amino acid substitutions, 154
M184I and D237E, were identified in the resultant variant (21). The double mutation 155
conferred moderate resistance to elvucitabine (about 10 fold) and cross-resistance to 156
lamivudine but not to other nucleoside inhibitors tested. Elvucitabine has also demonstrated 157
potent antiviral activity in HIV-infected patients with resistance to 3TC and other NRTIs. The 158
drug has good oral bioavailability and an intracellular half-life of >24 hours (15). 159
160
Apricitabine 161
Apricitabine (ATC) (Table 1) is a novel deoxycytidine NRTI currently in clinical 162
development for the treatment of HIV infection. In vitro selection for resistance with ATC 163
selected for M184V, V75I and K65R (25). The resulting mutants from this selection 164
conferred low-level resistance of less than 4 fold. Others showed that continuous passage of 165
HIV-1 already containing M184V, K65R or combinations of M41L, M184V and T215Y did 166
not result in any additional mutations (62). In vitro ATC has shown favorable antiviral 167
activity against HIV-1 wt strains and clinical isolates containing NRTI mutations including 168
M184V, L74V, and TAMs (11). ATC yielded virological response in treatment-naïve and 169
treatment-experienced HIV-1 infected patients whose viruses contained M184V and up to 5 170
TAMs. Resistance to ATC was reported to be slow to develop in vitro, and there is little 171
evidence of the development of resistance to this drug in patients (11, 25, 62). 172
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4’-Ethynyl-2-fluoro-2’-deoxyadenosine triphosphate 173
4’-Ethynyl-2-fluoro-2’-deoxyadenosine triphosphate (EFdA-TP) (Table 1) is a new NRTI, 174
now in pre-clinical development, that retains the 3’-OH group and has excellent antiviral 175
properties i.e EC50 of 0.07 nM against wt virus (31, 35) compared to approved NRTIs that 176
range in EC50 between 17-89 nM (38). This robust antiviral activity is due to a mechanism of 177
action that is different from approved NRTIs. Notably, EFdA-TP acts by binding through its 178
3’-primer terminus to RT; the addition of subsequent nucleotides is then prevented by 179
blocking the translocation of the primer strand on the viral polymerase (52). Thus, EFdA-TP 180
is termed a “translocation-defective reverse transcriptase inhibitor (TDRTI)”. Modeling 181
studies have confirmed the binding of EFdA-TP to a hydrophobic pocket of RT residues 182
consisting of A114, Y115, F160 and M184 and the aliphatic chain of D185 (52). 183
184
In an in vitro pre-steady-state kinetics study to determine toxicity, it was shown that EFdA-185
TP is a poor human mitochondrial DNA polymerase � (Pol �) substrate, suggesting that Pol 186
�-mediated toxicity might be minimal (82). Resistant variants including those containing the 187
K65R/L74V/Q151M complex did not affect the susceptibility of this compound while the 188
HIV-1 clone containing M184V alone conferred low to moderate level resistance to EFdA-189
TP (31). In vitro, a parental compound of EFdA-TP, 2’-deoxy-4’-C-ethynyl-adenosine (EdA) 190
selected for resistant variants after 58 passages with novel combination of mutations, 191
I142V/T165R/M184V (31). Site directed mutagenesis of clones containing the mutations 192
showed that either I142V or T165R alone did not affect the antiviral activity of EFdA-TP 193
while M184V alone or in combination with I142V or T165R demonstrated moderate 194
resistance to EFdA-TP. The triple mutant I142V/T165R/M184V had the highest resistance 195
amongst clones tested (31). 196
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GS-9131 198
GS-9131 (Table 1) is a prodrug of the nucleotide reverse transcriptase analogue GS-9148, 199
which belongs to the same family as TFV. GS-9131 demonstrated potent antiviral activity 200
against a variety of HIV-1 subtypes i.e. EC50 of 37 nM (12). In vitro, the parent drug (GS-201
9148) caused only low-level cytotoxicity compared to TFV (12). GS-9131 also demonstrated 202
synergy in combination with other antiretrovirals as well as potent antiviral activity against 203
multi-NRTI resistant strains, including K65R, M184V, and L74V, and only a minimal 204
increase in EC50 in regard to viruses carrying four or more TAMs (12). The use of GS-9131 205
was shown to result in 76-290 times more of the di-phosphorylated form of GS-9148 206
compared to GS-9148 itself (73). 207
208
GS-7340 209
GS-7340 (Table 1) is a prodrug of TFV that exhibits anti-HIV activity and that possesses a 210
favorable resistance profile. The EC50 of GS-7340 against HIV-1 in MT-2 cells was 0.005 211
µM compared to 5 µM for the parent drug TFV (40). In HIV-I infected patients, GS-7340 212
demonstrated enhanced antiviral activity, with no TFV mutations identified, and yielded 213
higher intracellular concentrations of TFV-diphosphate than did TFV itself (69). 214
215
CMX157 216
CMX157 (Table 1) is a lipid moiety prodrug of TFV that has activity against wt viruses of 217
major HIV-1 subtypes with EC50s ranging from 0.2 to 7.2 nM (38). In contrast TFV has 218
EC50s against HIV-1 group M and O that range between 500-2200 nM in PBMCs (24). In an 219
in vitro study, CMX157 demonstrated potent activity against NRTI-resistant strains, 220
including multidrug-resistant viruses against which it showed >300-fold activity compared to 221
TFV (38). The higher potency and lower EC50 of CMX157 is due to better cellular uptake 222
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than TFV and the fact that it is not a substrate for organic anion transporters. This permits it 223
to maintain high concentrations inside cells, in contrast to TFV that is actively metabolized 224
by organic anion transporters, leading to decreased intracellular concentrations (72). In vitro 225
and pre-clinical studies in rats showed that CMX157 also has a favorable cytotoxicity profile 226
in PBMCs and that it does not lead to nephrotoxicity as has been reported for TFV (38, 63). 227
No information is available on the selection of resistance mutations with CMX157. 228
229
Amdoxovir 230
Amdoxovir (AMDX) is a prodrug of β-D-dioxolane guanosine (DXG) that is currently in 231
phase II clinical trials (Table1). In vitro phenotypic analyses have shown that DXG is 232
effective against HIV-1 variants that are resistant to lamivudine and emtricitabine (M184V/I) 233
as well as against viruses that contain TAMs, while selection studies in MT-2 cells resulted in 234
the appearance of K65R and L74V (5, 51). The combination of AMDX together with 235
zidovudine (ZDV) in HIV-1 infected patients was shown to be synergistic, resulting in 236
reduced viral loads (57). AMDX thus represents a new NRTI that possesses potent antiviral 237
activity against NRTI-resistant viruses. 238
239
Festinavir (OBP-601) 240
Festinavir (OBP-601) (Table 1) is a new NRTI in the same family as stavudine (d4T) but that 241
has an improved safety profile. In vitro, it shows potent antiviral activity against wt HIV-1 of 242
multiple subtypes with EC50s ranging from 0.76-5.8 µM, compared to 1.57-6.06 µM for d4T 243
(90) . In a phenotypic susceptibility assay, viruses carrying the K65R and Q151M resistance 244
mutations were shown to be hypersusceptible to this compound. In contrast, a slightly 245
decreased antiviral response was observed against viruses carrying either TAMs or TAMs 246
together with K103N and M184V (90). More importantly, a strong synergistic effect of 247
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OBP-601 with several approved NRTIs and NNRTIs was observed against wt and resistant 248
viruses (90). 249
250
Newer NNRTIs 251
Etravirine 252
Etravirine (ETR) (Table 2), formerly known as TMC125, is a diarylpyrimidine (DAPY)-253
based NNRTI that possesses potent antiviral activity against both wt HIV-1 of multiple 254
subtypes as well as against some viruses containing NNRTIs resistance mutations (1, 88). 255
Specifically, ETR retains full activity against viruses containing the most prevalent NNRTI 256
mutation, K103N (1). In vitro, ETR is more difficult to generate resistance against compared 257
to initial NNRTIs (88). In clinical studies of ETR in combination with potent background 258
regimens that included NRTIs, integrase and protease inhibitors, it was observed that viral 259
loads became significantly decreased in patients with resistance to older NNRTIs and some 260
PIs (9, 45, 58). In vitro and clinical studies have described 20 ETR resistance-associated 261
mutations (RAMs) (V90I, A98G, L100I, K101E/H/P, V106I, E138A/K/G/Q, V179D/F/T, 262
Y181C/I/V, G190A/S, and M230L) and have allowed a weighted score to be assigned to each 263
mutation (3, 83, 89). Of these ETR RAMs, three or more are required for high level 264
resistance to occur, thus demonstrating a high genetic barrier to resistance compared to older 265
NNRTIs. The structure of ETR allows it to bind to the RT enzyme, such that mutations in the 266
NNRTI-binding pocket do not compromise binding and, thus, activity is maintained. ETR 267
can rotate within the pocket allowing multiple interactions despite the presence of mutations 268
in the binding pocket. (19). Because of its unique characteristics, ETR is the only NNRTI 269
approved for treatment of NNRTI experienced patients. 270
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Only a few studies have prospectively studied the efficacy of ETR in combination with other 272
background regimens in NNRTI experienced patients (30, 49). In the phase III DUET-1 and 273
DUET-2 studies, 57% of patients in the ETR arm versus 36% in the placebo had a viral load 274
<50 copies/ml after 96 weeks of treatment (30). 275
In the DUET-1 and DUET-2 clinical studies, it was found that patients who experienced 276
virologic failure had greater numbers of ETR resistance mutations at baseline than treatment 277
successes. Second, patients who experienced virologic failure were often found to have 278
received background regimens that were less potent than the drugs given to patients who did 279
not fail therapy (84). In this study, the V179F, V179I and Y181C mutations in RT were 280
commonly associated with treatment failure alongside changes at positions K101 and E138 281
(84). The authors concluded that these mutations usually emerge in a background of other 282
multiple NNRTI mutations and were, in most cases, associated with a decrease in phenotypic 283
sensitivity to ETR. 284
285
Another sub-analysis of the DUET trial studied the impact of background regimen on 286
virologic response to ETR, and the authors further confirmed that a higher virologic response 287
rate was observed in patients who demonstrated an increased activity of the background 288
regimen, with the highest responses being achieved in patients who used more than two 289
active agents in addition to ETR (85). In the TMC125-C227 (ETR) trial, ETR was inferior to 290
a protease inhibitor (PI) in PI-naïve patients with a history of previous NNRTI failure (78). In 291
a post-hoc analysis of baseline resistance data for the TMC125-C227 trial, a diminished 292
virological response was observed in patients who possessed the following characteristics at 293
baseline; the presence of Y181C, a baseline ETR fold change of ≥ 10, and a higher number of 294
ETR resistance mutations (78). In another study, the authors studied 42 NNRTI treatment-295
experienced patients for 6 months on an ETR containing regimen (49). At failure, 12 of 42 296
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patients developed at least one new NNRTI mutation. The most frequently selected mutations 297
included V179I, Y181C and V179F. 298
299
In contrast, several studies have researched the theoretical potential of ETR, based on the 300
resistance patterns of patients who previously failed NNRTI therapy and accumulated ETR 301
RAMs. These studies have observed a prevalence of more than three ETR RAMs among viral 302
isolates from patients experiencing NNRTI treatment failure, ranging from 4.6% to 10%, 303
while the prevalence of isolates with single ETR RAMs was 17.4% to 35.9% (41, 61, 70, 71). 304
These studies concluded that there is a low prevalence of ETR resistance at baseline and that 305
patients with prior failure to NNRTIs could potentially benefit from ETR rescue therapy. 306
However, these analyses focused on patients in developed countries that have full access to 307
the most potent antiretroviral drugs and patients are constantly monitored for viral load and 308
the development of resistance. 309
310
In contrast, patients in countries with limited resources developed resistance faster due to a 311
lack of potent antiretroviral drugs and drug resistance testing. Some studies in resource 312
limited settings have observed a high prevalence of NNRTI resistance mutations associated 313
with ETR resistance among patients failing an NNRTI containing regimen (32, 34, 39). Using 314
NVP in the failing regimen was associated with intermediate and reduced response to ETR 315
while use of EFV and co-administration of 3TC reduced the risk of ETR resistance (39). The 316
authors concluded that the frequent occurrence of NNRTI mutations in resource limited 317
settings in which drug resistance testing is rare might compromise the continuous use of ETR 318
and also its use in second line therapy. The Y181C mutation, associated with NVP therapy, 319
has been reported with a high prevalence in NNRTI experienced patients and shown to 320
decrease susceptibility to ETR (39, 41, 46, 47). This demonstrates cross-resistance of both 321
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NVP and ETR as confirmed by our group (3). Thus, the wide spread use of NVP in resource 322
limited settings without resistance testing casts doubt as to whether ETR could be effective in 323
NNRTI experienced patients in poorer countries. One of these studies suggested that ETR 324
should be avoided in salvage regimens in the setting of first-line NVP failure where drug 325
resistance testing is not performed (47). Another study analyzed the prevalence of minority 326
variants in treatment-naïve and NNRTI experienced patients by ultradeep pyrosequencing: 327
while such variants were not identified in any of 13 drug-naive patients, it was shown that 7 328
of 20 patients who had failed an NNRTI-containing regimen possessed minority variants as 329
well as ETR associated-NNRTI resistance mutations (86). This suggests that minority 330
variants in NNRTI experienced patients may lead to decreased ETR activity and to virologic 331
failure (66). 332
333
In vitro selection and clinical trials results with ETR have identified amino acid substitutions 334
at position 138 in RT (3, 83). Mutations at position 138 are not associated with resistance to 335
older NNRTIs. Although these mutations conferred only low-level resistance to ETR, E138K 336
is also associated with resistance to most newer NNRTIs (Table 2); its emergence may also 337
facilitate the development of additional ETR mutations (3). The connection domain mutation 338
N348I has been identified and implicated in reduced susceptibility to NVP and EFV as well 339
as to the nucleoside analogue ZDV (26). Two independent studies have assessed and 340
confirmed that this mutation also decreases susceptibility to ETR, either alone or in 341
combination (27, 50). This effect was reversed when M184V was co-expressed with N348I. 342
Additional connection domain mutations found to be associated with impaired susceptibility 343
to ETR were T369I and E399G. 344
345
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Two independent genotypic scores have been established to predict ETR resistance. The first, 346
developed by Janssen, has been correlated with treatment response in the DUET studies and, 347
when combined with in vitro selection studies, identified 17 (recently updated to 20) ETR 348
RAMs (83, 89). In this analysis, any three or more of these mutations were required to cause 349
resistance to ETR. A second score by Monogram is based on a correlation of phenotype and 350
genotype results of 4,923 samples containing at least one NNRTI mutation (54). The 351
Monogram genotypic score identified 30 mutations associated with ETR resistance with the 352
weighted score for each mutation being slightly higher than the 20 mutation-based Janssen 353
score. However, the final interpretation of the two scores seems to be similar. 354
355
Rilpivirine (TMC278) 356
Rilpivirine (RPV) (Table 2), also known as TMC278, is another DAPY compound that was 357
recently approved for treatment of NNRTI-naïve patients. The structure and binding of RPV 358
in the NNRTI binding pocket is similar to that of ETR, which allows reorientation of both 359
compounds within RT. In vitro, RPV possesses subnanomolar activity against wt HIV-1 of 360
multiple subtypes and shows antiviral activity against viruses containing many NNRTI 361
resistance-associated mutations (4). NNRTI RAMs emerging in culture under RPV selective 362
pressure included combinations of V90I, L100I, K101E, V106A/I, V108I, E138G/K/Q/R, 363
V179F/I, Y181C/I, V189I, G190E, H221Y, F227C, and M230I/L. The resistance profile and 364
genetic barrier to the development of resistance of RPV are comparable to those of ETR. 365
High-resolution crystal structures of RPV in complex with HIV-1 RT reveal that the 366
cyanovinyl group of TMC278 is positioned in a hydrophobic tunnel connecting the NNRTI-367
binding pocket to the nucleic acid-binding cleft (18). Both RPV and ETR exhibit similar 368
flexibility in adapting to resistance mutations. In the ECHO and THRIVE phase 3 trials (14, 369
55), resistance analysis showed a slightly higher proportion of treatment failures in the RPV 370
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arm compared to the EFV arm. The most frequent NNRTI mutation in the RPV arm was 371
E138K in addition to mutations such as Y181C, K101E, H221Y, V901, E138Q, and V189I 372
(76). Also, the proportion of NRTI mutations that emerged in the study was higher in the 373
RPV arm than the EFV arm. The NRTI mutations selected included M184I/V, K65R, K219E 374
and Y115F. 375
376
The IAS-USA recently published a total of 15 mutations (K101E/P, E138A/G/K/Q/R, 377
V179L, Y181C/I/V, H221Y, F227C, and M230I/L) associated with decreased susceptibility 378
to RPV (29). These mutations have been described based on in vitro studies and in patients in 379
whom RPV was failing. The quantitative impact of each of these mutations on RPV 380
resistance differs. 381
382
Dapivirine (TMC 120) 383
Dapivirine (TMC 120) (Table 2) is another DAPY compound that can accommodate some 384
mutations within the NNRTI binding site without significant loss of activity (42, 43). TMC 385
120 has shown potent antiviral activity against both wt and NNRTI-resistant HIV-1 strains 386
(22, 79). In 2004, Janssen officially licensed the further development of TMC 120 for use as 387
a vaginal microbicide to the International Partnership for Microbicides (IPM) to help prevent 388
sexual transmission of HIV-1. 389
390
The results of both phase I and II studies have shown that TMC 120 was widely distributed 391
through the lower genital tract with low systemic absorption when administered as a vaginal 392
gel formulation for up to 42 days (59, 60). The gel was safe and well tolerated. In vitro 393
selection studies have identified drug resistance mutations in the presence of TMC 120, 394
notably L100I, K101E, V108I, E138K/Q, V179M/E, Y181C and F227Y (80, 81). Most of 395
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these TMC 120 resistance-associated mutations occur at exactly the same position as many of 396
the mutations associated with ETR and RPV resistance (3, 4, 89). However, in one of these 397
studies, it was shown that sub-optimal concentrations of TMC120 alone facilitated the 398
emergence of common NNRTI resistance mutations while sub-optimal concentrations of 399
TMC120 plus tenofovir (TFV) gave rise to fewer mutations (80). Due to the likelihood of 400
transmitted resistant strains in HIV-1 infected individuals, resistance mutations might impact 401
the ability of a single drug in preventing HIV-1 as a microbicide. Using a combination of 402
antiviral drugs of different classes may be useful. Another study showed in an in vitro model 403
that using TMC 120 in combination with TFV as a microbicide was more potent and 404
exhibited synergy in comparison with either drug alone (79). 405
406
Lersivirine 407
Lersivirine (Table 2) is a new NNRTI belonging to the pyrazole family and is being 408
developed by Pfizer. In an in vitro resistance study, lersivirine selected for the amino acid 409
substitutions; V108I, E138K, V179D, F227L and M230I (16). In a phase II b trial, Pozniak 410
and colleagues reported better responses in patients with EFV compared to lersivirine, i.e. 411
86% versus 79% respectively (87). Amongst patients who failed lersivirine, the mutations 412
identified included K101E, V106M, V108I, H221Y, Y188H, F227C/L and L234I. 413
414
RDEA806 415
RDEA806 (Table 2) is a novel NNRTI being developed by Ardea Biosciences. In a genotypic 416
and phenotypic analyses of mutant viruses selected by RDEA806, the K104E, E138K, T240I, 417
V179D and F227L substitutions were identified (91). Phenotypic analysis of these mutations 418
demonstrated that RDEA806 requires at least 3 mutations for greater than 10 fold loss of 419
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susceptibility. In a phase 2a trial, RDEA806 was well tolerated and exhibited robust antiviral 420
activity with no genotypic or phenotypic changes (56). 421
422
Conclusion 423
An ideal new HIV inhibitor should possess a high genetic barrier in regard to potential 424
development of resistance for this same drug class as well as a unique resistance profile in 425
both B and HIV-1 non-B subtypes. NRTI resistance mutations are widespread and there is 426
currently no approved NRTI compound that possesses activity against all NRTI mutations. 427
However, a number of new NRTIs with potent activity against NRTI-resistant viruses are 428
now in preclinical and clinical development. In some cases, these compounds possess the 429
ability to maintain high concentrations inside cells (e.g. CMX157, GS-9131 and GS-7340) or 430
may have a different mechanism of action than older NRTIs, such seems to be the case for 431
EFdA-TP. Although M184V is a common NRTI mutation that confers high-level resistance 432
to 3TC and FTC and cross-resistance to other NRTIs, the levels of resistance conferred 433
against newer NRTIs such as ELV, ATC, and EFdA-TP is low-level, suggesting that these 434
compounds may be useful against M184V-containing viruses. It will be important to 435
determine whether selection of M184V in patients receiving ELV, ATC or EFdA-TP may 436
result in elevated viral loads and treatment failure. Hopefully, their potential to synergize 437
either together or with currently approved drugs will make them important components of 438
future antiretroviral strategies. 439
440
ETR is the only NNRTI approved for treatment of HIV-1 NNRTI experienced patients. In a 441
limited number of studies, ETR has proven to be effective in patients with treatment failure 442
who harbor viruses that are resistant to initial NNRTIs and that carry several resistance 443
mutations. A number of studies have shown that patients who fail NVP therapy are more 444
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prone to develop ETR resistance mutations faster than those who fail EFV therapy. 445
Considering the wide-spread use of NVP in developing countries, the use of ETR in NNRTI 446
experienced patients in these settings is under debate. RPV that is now approved for 447
treatment naïve patients shows high level cross-resistance with ETR. Therefore, the use of 448
RPV in first-line therapy may jeopardize the future of ETR as a second line NNRTI and the 449
sequential use of these drugs is not recommended. The emergence of E138K as a signature 450
mutation for almost all new NNRTIs (both approved and under clinical development) is 451
another limitation of these compounds. 452
TMC120, Lersivirine and RDEA806 are in still in phase II clinical trial and large scale phase 453
III trials are required to exploit their potential use in the clinics. Some mutations have been 454
selected in the presence of these three compounds together with ETR and RPV. The fact that 455
TMC120 in combination with TFV as a microbicide is more potent and decreases the 456
possibility of selecting for resistance mutations than either compound alone shows potential 457
for such an antiviral based microbicide in preventing HIV-1 infection. The search for novel 458
NNRTIs should now focus on compounds with different resistance profiles, so as to broaden 459
treatment options for patients who have experienced NNRTI therapy failure. Overall, new 460
NRTI and NNRTI agents can provide a welcome therapy option for patients with existing 461
NRTI or NNRTI resistance and also for patients who are naïve to therapy. 462
463
ACKNOWLEDGMENTS 464
Research in our laboratories is supported by grants from the Canadian Institutes of Health 465
Research and the Réseau FRSQ-SIDAMI. E.L.A. is the recipient of a Departmental 466
Scholarship from the Département de Microbiologie et d’Immunologie, Université de 467
Montréal. CT is the Pfizer/Université de Montréal Chair on HIV translational Research. We 468
thank Peter Quashie for helping to generate the structures of the compounds.469
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470
471 472 473 474 475 476 477
Table 1: New NRTIs undergoing pre-clinical or clinical development
Drug Candidate ELV ATC EFdA-TP GS-9131
Chemical Structure
Phase of Development
IIb IIb Pre-clinical development I
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478 Table 1: Cont.
Drug Candidate GS-7340 CMX157 Amdoxovir Festinavir
Chemical Structure
Phase of Development
II II II II
479 480 481 482 483 484 485 486 487 488
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489 Table 2: New NNRTIs that approved or are undergoing clinical development.
Drug candidate
ETR RPV TMC 120 RDEA806
Lersivirine (UK-453061)
Chemical structure
Phase of development
Approved (2008) Approved (2011) License for HIV-1 microbicide development
IIb IIb
490
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