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Antiviral Drug Resistance and the Need for Development of New HIV-1 Reverse 1

Transcriptase Inhibitors. 2

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Eugene L. Asahchop1, 2, 3, Mark A. Wainberg1*, Richard D. Sloan1 and Cécile L. 4

Tremblay2, 3. 5

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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

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*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

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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

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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

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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

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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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