HIV Drug Resistance and the Advent of Integrase ... - Springer Link

Curr Infect Dis Rep (2013) 15:85–100 DOI 10.1007/s11908-012-0305-1

HIV/AIDS (R MACARTHUR, SECTION EDITOR)

HIV Drug Resistance and the Advent of Integrase Inhibitors Peter K. Quashie & Thibault Mesplède & Mark A. Wainberg

Published online: 25 November 2012 # Springer Science+Business Media New York 2012

Abstract This review focuses on the topic of HIV integrase inhibitors that are potent antiretroviral drugs that efficiently decrease viral load in patients. However, emergence of resistance mutations against this new class of drugs represents a threat to their long-term efficacy. Here, we provide new information about the most recent mutations identified and other mutations that confer resistance to several integrase inhibitors, such as new resistance mutations—for example, G118R, R263K, and S153Y—that have been identified through in vitro selection studies with second-generation integrase strand transfer inhibitors (INSTIs). These add to the three main resistance pathways involving mutations at positions Y143, N155, and Q148. Deep sequencing, structural modeling, and biochemical analyses are methods that currently help in the understanding of the mechanisms of resistance conferred by these mutations. Although the new resistance mutations appear to confer only low levels of cross-resistance to second-generation drugs, the Q148 pathway with numerous secondary mutations has the potential to significantly decrease susceptibility to all drugs of the INSTI family of compounds. P. K. Quashie : T. Mesplède : M. A. Wainberg McGill University AIDS Centre, Lady Davis for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada P. K. Quashie : M. A. Wainberg Division of Experimental Medicine, Faculty of Medicine, McGill University, Montreal, Quebec, Canada M. A. Wainberg Department of Microbiology and Immunology, Faculty of Medicine, McGill University, Montreal, Quebec, Canada M. A. Wainberg (*) McGill University AIDS Centre, Jewish General Hospital, 3755 Cote Sainte Catherine, Montreal, QC H3T 1E2, Canada e-mail: [email protected]

Keywords HIV-1 . Integrase strand transfer inhibitors (INSTIs) . Resistance . Mutations

Introduction Although current highly active antiretroviral therapy (HAART) typically includes a combination of two reverse transcriptase inhibitors and a protease inhibitor or a nonnucleoside reverse-transcriptase inhibitor, drug resistance threatens the long-term efficacy of HAART. The problem of resistance in HIV therapy has been reviewed elsewhere [1, 2]. In spite of the fact that fewer patients than previously in western countries are experiencing either treatment failure or drug resistance, the topic remains vital in developing country settings. Against this background, the addition of new classes of drugs is important to limiting the emergence of resistant strains, and the latest class of antiretroviral (ARV) drugs approved for treatment of HIV infection is integrase strand transfer inhibitors (INSTIs). The integration process is a well-known two-step reaction catalyzed by the retroviral protein integrase (IN; reviewed in [3, 4]). Both reactions (3′ processing and strand transfer) require the coordination of divalent ions (Mg2+ or Mn2+) by a catalytic triad consisting of amino acids D64, D116, and E152. INSTIs bind to IN and specifically target the strand transfer step of integration [5, 6]. These drugs have demonstrated their efficacy against HIV in vitro and in patients and are particularly useful against viral strains that are resistant to other drug classes [7]. They are active against both B- and non-B HIV subtypes both in tissue culture and in patients [8, 9]. The prototypic INSTI, raltegravir (RAL) [10, 11•] was approved for therapy in 2007, while elvitegravir (EVG) [12] and dolutegravir (DTG) [13] are now in advanced clinical trials. INSTIs were found to be highly potent in the clinic; their use resulted in faster declines in

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viral load upon treatment initiation than had previously been observed, and this was attributed to the ability of INSTIs to target a later stage in the HIV replication cycle than other drug classes, rather than to greater antiviral activity on their part. Resistance pathways against first-generation INSTIs were identified whose primary mutations include the substitutions N155H, Q148K/R/H, and Y143R/C [8, 14–16]. Second-generation INSTIs, such as MK-2048 [17–20] and DTG [17, 19–24], possess a more robust resistance profile than do RAL and EVG [19, 20, 24–26]. Several other compounds are currently in early development, such as compound G [20]. This review will focus on resistance to RAL, EVG, DTG, and MK-2048.

Second-Generation INSTIs and Drug Resistance Several resistance mutations have been identified in regard to second-generation INSTIs (Table 1). Although some of these mutations were previously observed with firstgeneration drugs, most of them are still poorly characterized. Initial in vitro selection studies with DTG have led to the identification of mutations T124A, S153F/Y, and L101I [24, 27]. However, experiments with viruses containing these mutations showed that none of them, alone or in combination, significantly decreased DTG susceptibility. When similar selections were undertaken with viruses containing either Q148R or Q148H at baseline, numerous additional mutations were identified, among which E138K and G140S were the most common [27]. Importantly, even the combination of five of these mutations did not decrease drug susceptibility to DTG by more than approximately 20-fold in this study [27]. In other in vitro selection experiments, additional mutations were identified in subtype B, C, and recombinant A/G viruses [28•]. The R263K mutation was the most common mutation identified in this last study and conferred low-level resistance to DTG, as well as a decrease in viral fitness and strand transfer activity in vitro, due to a reduction in DNA binding [28•]. Additional mutations H51Y, S153Y, and G118R were identified in the same study [28•]. Similar in vitro selection studies with MK-2048 led to the appearance of G118R and the secondary mutations E138K [19], G140E [25], N155H, and S153Y [17].

Activity of INSTIs Recent resolution of the structure of the prototype foamy virus PFV intasome has been broadly used to explore the mechanisms of action of INSTIs and their resistance mutations [6, 29–32]. INSTIs bind to the catalytic core domain of IN and compete with host DNA binding [33]. These drugs contain a halogenated phenyl group that invades the

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catalytic pocket and displaces the 3′ viral end [30, 34] and three coplanar oxygen atoms that chelate the divalent ions within the catalytic pocket, thus inhibiting the activity of the catalytic DDE triad [4, 6, 26, 35–37]. Although coordination of these ions by the triad is also necessary for 3′ processing, INSTIs are specific for strand transfer and only poorly inhibit 3′ processing [5, 33]. This is due to an allosteric hindrance between the halogenated phenyl group and the 3′ dinucleotide cleaved during 3′ processing that prevents efficient binding of INSTIs before 3′ processing occurs [30, 34]. The fact that all early IN inhibitors (INIs) target the catalytic core domain is an argument for the development of alternative strategies to block integration, such as allosteric INIs (ALLINIs). Not surprisingly, most INSTI resistance mutations lie in the catalytic core domain surrounding the catalytic pocket that contains the DDE triad (Fig. 1). Whereas Y143 is slightly outside the active site, D64 and E152 surround N155, and Q148 lies at the core of the triad, in close proximity with all three acidic residues of the triad. In addition, Q148 interacts with the viral DNA 5′ end [38]. The G118 residue is highly conserved and is in close proximity with a key residue of the triad, D116. Similarly, S153 is next to E152. In contrast, R263K is an unusual mutation at the C-terminal end of the protein. R263K is not within the catalytic pocket but contributes to viral DNA binding [28•] and might be important for IN nuclear import [39].

Resistance Mechanisms In view of their strategic locations, the Q148H and N155H mutations are thought to trigger conformational changes within the catalytic pocket that result in an increase in the binding energy of INSTIs [34]. In contrast to these two residues that do not make direct contact with INSTIs, Y143 is thought to connect to RAL by pi stacking between the tyrosine phenol ring and the RAL 1,3,4 oxadiazole group [34]. EVG, MK-2048, and DTG do not have this oxadiazole group and are largely resistant to mutations at position Y143 [20, 40]. G118R may cause changes in the geometry of the catalytic triad, seemingly decreasing the ability of second-generation INSTIs to chelate divalent ions within the catalytic core [19]. Alternatively, this mutation could decrease drug binding by steric hindrance [26]. The mechanism of resistance of the DTG-associated resistance mutation R263K is not well characterized but could be linked to decreases in viral DNA binding, as described previously [28•]. Important results have emerged through measurements of the RAL, EVG, and DTG dissociation rate constants [41] from an integrase–DNA complex containing IN proteins bearing resistance mutations. These studies demonstrated a striking correlation between dissociation rate and levels of resistance [42•]. Both the Q148H and

Curr Infect Dis Rep (2013) 15:85–100 Table 1 Susceptibilities of common drug resistance mutations to several INSTIs expressed as fold change (FC) in susceptibility of mutant virus or integrase protein relative to wild type

FC scores less than 2 are designated as (−), while those of 2– 10, 10–20, 20–50, 50–100 and >100 are designated +, ++, +++, ++++, and +++++, respectively. ND 0 not done. Data are derived from multiple sources [17, 19, 20, 24, 26, 28•, 40, 45, 56, 175, 176], as well as from the Stanford HIV Drug Resistance Database [177]

Mutations

87

Fold change (FC) RAL

EVG

DTG

MK-2048

T66RK L74M E92Q T97A G118R E138K G140S Y143C Y143R Q148H

+ − + + + + + + +++ ++

++++ − +++ + + + − − +

− − + − + + − − − −

− ND − ND + + + + + +

Q148K Q148R N155H R263K T66I/R263K L74M/G118R L74M/N155H E92Q/N155H T97A/Y143C T97A/Y143R G118R/E138K

++++ +++ +++ − − +++ +++ ++++ +++++ +++++ +

++++ ++++ +++ + +++++ + +++ +++++ − − −

− − − ++ ND ND − + − − +

− + + − ND ND ND + ND ND ++

E138K/Q148H E138K/Q148K E138K/Q148R G140S/Q148H G140S/Q148K G140S/Q148R L74M/T97A/Y143G L74M/T97A/E138A/Y143C

+ +++++ +++++ +++++ + +++++ +++ ++

++ +++++ +++++ +++++ ++++ +++++ ND ND

− ++ + ++ − + − −

ND ND ND ++ + ND ND ND

E138A/G140S/Y143H/Q148H

+++++

ND

+++

ND

N155H resistance mutations increased the DTG koff by 13.7and 7.4-fold, respectively, whereas the Y143 mutations had little effect [42•]. The same study showed that both Q148H and N155H significantly increased the koff for RAL and EVG but that Y143C/H/R affected only RAL but not EVG dissociation from IN–DNA [42•]. This result is in agreement with the absence of resistance to EVG by the Y143R mutation [40]. This suggests that the three main pathways of resistance to INSTIs act, at least in part, by reducing the residence time of the drugs within IN. Indeed, the possible association between slow dissociation rate and the development of resistance has been documented in other studies [42•, 43]. Dissociation rate is therefore an important factor measuring the quality of a drug [43] and should be taken into account in the development of future INIs, since INSTIs bind with high affinity to the IN enzyme and dissociation is

a slow process that is sometimes almost irreversible. Since the IN enzyme is packaged within the viral particle and is delivered to the infected cell as a single dose, this excludes the possibility that new IN target structures will be synthesized prior to integration. However, resistance mutations that alter IN–DNA binding activity, stability, or localization of the preintegration complex might impact the activity of the INSTIs, and this field requires further investigation.

Should We Test for Resistance? Drug resistance is often tested phenotypically in cell culture or biochemically using recombinant purified IN proteins. Resistance can be tested in tissue culture selection studies by sequencing the IN region of the pol gene of the viral

CCD

D 288

NTD

Q 213

Curr Infect Dis Rep (2013) 15:85–100 M 50

L1

88

CTD

DTG

G 193

++++++ +++++ ++++ +++ ++ + -

MK-2048

++++++ +++++ ++++ +++ ++ + -

EVG

++++++ +++++ ++++ +++ ++ + -

RAL

E 138 G 140 Y 143 Q 148 E 152 N 155 E 157

D 116 G 118

E 92 Q 95

D 64

H 51

++++++ +++++ ++++ +++ ++ + -

R 263

T 66

P 145 S 147 V 151 S 153

*

Fig. 1 Map of HIV-1 integrase mutations showing residues prone to mutate into resistance mutations and associated levels of resistance/ susceptibility. For clarity, each position associated with resistance is labeled only once, and the drug that was first reported to select for a particular mutation is stipulated. Key active site residues are indicated in blue. Levels of resistance shown in the figure as indicated by fold change (FC) relative to wild type are as follows: FC values 20 are designated +, ++, +++, ++++, +++++, and ++++++, respectively. NTD, N-terminal domain; CCD, catatlytic core domain; CTD, Cterminal domain. Data were obtained from [17, 19, 20, 24, 26, 28•, 40, 45, 56, 175, 176], as well as from the Stanford HIV Drug Resistance Database [177]

genome after increasing drug concentration. Alternatively, viruses that are obtained from patients can be sequenced after treatment failure. There is usually a good correlation between mutations selected in culture and those detected in patients. In preclinical studies, tissue culture selections are performed by infecting cells in the presence of suboptimal concentration of inhibitor [44]. The choice of cells used during these selections can impact on the development of resistance mutations. For example, selection with DTG in MT-2 cells resulted in the emergence of T124A, S153F/Y, and L101I in subtype B virus, whereas similar experiments in cord blood monocytic cells yielded the R263K mutation [28•]. The stochastic emergence of resistance mutations can sometimes also be demonstrated [25, 45–47]. In addition, no

study has demonstrated that a particular cell model is better than others for selecting clinically relevant resistance mutations. This underlines the importance of performing multiple in vitro selection studies using different types of cells. More recently, new approaches such as ultradeep sequencing to assess the development of resistance against INIs in patients have been developed [48]. Recently, 23 patients receiving RAL-based salvage therapy were monitored longitudinally for the emergence of resistance mutations [49]. This study confirmed the role of the three main resistance pathways Y143, N155, and Q148 for RAL resistance and the potency of ultradeep sequencing [48]. Studies on the evolution of resistance mutations in patients infected with viral strains resistant to multiple classes of drugs and

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treated with EVG showed that IN resistance mutations identified during early treatment failure were often absent from later time points because they had been substituted by other mutations [50]. Similar observations have been made with RAL [48, 51–53], and the development of resistance through the cumulative acquisition of mutations has also been described for this drug [48, 49, 51–53]. Thus, longitudinal genotyping of viral samples from patients undergoing INI treatment should be implemented when possible. This will improve our understanding of the emergence and dynamics of resistance mutations associated with INSTIs. In regard to both RAL and EVG, Q148 mutations can emerge and substitute for initial mutations at Y143 and N155 [17, 45, 48, 50, 54]. The emergence of Q148 in the presence of alternative resistance pathways seems to be due to increased viral replication fitness associated with the latter mutation. This is of concern in regard to potential cross-resistance against second-generation INSTIs, as well as first-generation compounds such as EVG and RAL, and highlights the need for genotyping and resistance testing for patients failing RALor EVG-based therapy, particularly in the case of mutations at position 148.

Cross-Resistance Notwithstanding that second-generation inhibitors have been developed to avoid cross-resistance with firstgeneration drugs, they bind to the same catalytic site and possess similar chemical properties as first-generation INSTIs. Multiple cross-resistance mutations have been described for the first-generation drugs RAL and EVG (reviewed in [55] and [56]). Among the three main resistance pathways, a notable exception is Y143H/R, which is specific for RAL and does not compromise the activity of EVG [40]. However, the addition of other secondary mutations to Y143 mutations can confer up to 66-fold resistance to EVG [20]. The two other pathways, N155 and Q148, confer cross-resistance against both EVG and RAL [55, 56]. Despite having been originally identified during tissue culture selection studies with the second-generation INSTI MK-2048 [19], G118R was later identified in a patient with a CRF02_AG infection who failed treatment with RAL [57]. In addition, G118R was shown to confer resistance to MK2048, RAL, and DTG [19, 26, 57]. The reason that G118R remains uncommon in patients treated with RAL might be its severe impact on viral fitness [19]. The secondary mutation E138K that augments levels of MK-2048 resistance only partially restores viral fitness in viruses that contain G118R [19]. S153Y is another mutation that confers lowlevel cross-resistance to both MK-2048 and DTG [17, 24, 27, 28•, 58]. Another second-generation INSTI resistance mutation, R263K, was originally identified as a secondary

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mutation to T66I during selection studies with EVG, and R263K confers ~5-fold resistance against EVG [46, 59] but not against RAL [59], as was also observed in a biochemical strand transfer assay [28•]. Thus, some degree of crossresistance exists between the first- and second-generation INSTIs, but most of the mutations responsible seem to confer only low- or modest-level resistance to the secondgeneration compounds. Despite this, mutations emerging from the Q148 pathway seem to be more potent against the latter drugs, and Q148K/ E138K, Q148R/N155H, and Q148R/G140S confer 19-, 10-, and 8-fold resistance, respectively, to DTG [24]. The addition of further secondary mutations within the Q148HR/ G140S pathway can lead to as much as 27-fold resistance to DTG [60]. Similarly, the Q148H/G140S mutations can decrease susceptibility to MK-2048 by 12-fold [20], and mutations at position Q148 with multiple secondary mutations can result in up to 250-fold resistance to this drug [17, 25]. These data indicate that mutations that emerge from the Q148 pathway can confer cross-resistance to secondgeneration INSTIs, in contrast with emergence of the N155 and Y143 mutations [20]. Early detection of drug resistance can aid in regimen modification, thus preventing the development of viruses that might be completely resistant to the INSTI class of inhibitors.

Related Findings Tissue culture selection studies have identified several mutations that reduce HIV-1 susceptibility to DTG and MK2048. Resistance pathways involving mutations at positions G118, R263, S153, N155, and Q148 can limit secondgeneration INSTI activity. However, only resistance mutations emerging from the Q148 pathway seem to confer a significant (i.e., >10-fold) decrease in susceptibility to these compounds. Importantly, second-generation INSTIs have proven to be robust against the emergence of resistance, as shown in the phase IIb SPRING clinical studies with DTG, for which no resistance mutation has yet been identified in patients after 96 weeks of treatment [61, 62]. In addition, DTG has been shown to be active against viruses resistant to RAL [63, 64], although viruses bearing Q148 mutations together with several secondary mutations can necessitate an increase in the clinical dose of DTG [63, 64]. Clearly, the hope is that second-generation INSTIs will be able to target viruses that are resistant to first-generation drugs. The emergence of mutations at position Q148 should be monitored carefully during therapy, due to the possible emergence of novel resistance pathways. Alternative strategies that target integration but that are not based on the catalytic triad—for example, ALLINIs—are promising, and such compounds retain full activity against INSTI-resistant viruses.

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First-Generation Integrase Inhibitors The IN of HIV is pivotal in the viral replication cycle as it catalyzes the insertion of the reverse transcribed viral genome into host chromatin. This unique process has always been considered a viable drug target that several early studies attempted to exploit [65]. Early INIs included peptides [66, 67], nucleotides [68], and DNA complexes [69], as well as small molecules derived either from natural products [68] or by rational drug design strategies [67, 70]. Even though some of these compounds advanced into preclinical trials, further clinical development was always curtailed due to in vivo toxicity and/or nonspecific off-target effects. More detailed reviews on the development of early INIs are available [65, 67, 71]. In order for any inhibitor to be considered useful as an antiviral in combination therapy for HIV, selectivity (such as for IN), which is distinct from effects on other targets (such as RT and protease), needs to be proven. The 4-aryl-2,4diketobutanoic acid inhibitors containing a distinct diketo acid (DKA) moiety were identified in 2000 by Merck investigators from a screen of 250,000 compounds and, for a time, were the only biologically validated INIs [72••]. Their antiviral activity in cell culture was mitigated by the development of resistance mutations in the IN protein, thereby confirming their mode of action [72••]. These compounds, exemplified by L-731988 [73], were found to inhibit strand transfer with much higher potency (IC50080 nM) than 3′ prime processing (6 μM) [72••], and they were thus referred to as strand transfer inhibitors (STIs). IN, like most phosphotransferase enzymes, requires two divalent cations bound at the active site for activity; Mg2+ is likely used in vivo, although Mn2+ is used in some in vitro assays [74]. Most STIs that have been described, including DKA compounds, inhibit IN by chelation of bound cations in a dosedependent manner [75].

Early Clinically Approved Integrase Inhibitors Raltegravir The study of lead compounds including L-31988 and L870812 by Merck pharmaceuticals led to the development of Raltegravir (RAL; Issentris®, MK-0518) (Fig. 1), which in 2007 became the first INI (and currently only) approved for treatment in both ARV naive and treatment-experienced patients [76]. RAL [76] was shown in multiple trials, such as BENCHMRK, to achieve efficient viral load suppression in ARV-experienced patients when included in an optimized background ARV regimen [77]. In the BENCHMRK trials, 57 % of patients achieved plasma levels of HIV-1 RNA 5) to INIs have shown that RAL has a modest genetic barrier to resistance development. As was stated above, three major resistance pathways involving nonpolymorphic residues have been extensively described and characterized for RAL: E92QV/N155H, T97A/Y143CHR, and G140CS/ Q148HKR [85, 86]. Although these three pathways have been shown to arise separately, some recent reports suggest that they may be linked. The G140S/C and E92Q/V mutations by themselves impart greater than 5- to 10-fold resistance to RAL [87] but usually appear only after the N155H and Q148HKR mutations [88], leading to FC >100 for the combined mutations. In addition to these major resistance mutations, several polymorphic and nonpolymorphic residues have been identified that impart 90 % in the arm who did not switch (n0 354). Thus, this study failed to establish noninferiority of RAL to LPV in the treatment of ARV-experienced HIVpositive individuals with undetectable viremia [93]. Of the 11 patients who experienced virologic failure with HIV-1 RNA levels>400 copies/mL, 8 harbored RAL-resistance substitutions [94]. Elvitegravir Elvitegravir (GS-9137) (EVG) (Fig. 1) has recently been approved by the FDA. It is not a DKA but a mono-keto acid resulting from early modification of the DKA motif by the Japan Tobacco Company [95]. This work resulted in a group of 4-quinolone-3-glyoxylic acids, all of which had a single pair of coplanar ketone and carboxylic groups and retained high specificity for and efficacy against the strand transfer reaction similar to DKA compounds [96]. EVG, now being developed by Gilead Sciences, has been shown to have an in vitro IC50 of 7 nM against IN and an antiviral EC90 of 1.7 nM when assayed in the presence of normal human serum [97]. EVG displayed ~30 % bioavailability in dogs and rats with maximal plasma concentrations being achieved in 0.5–1 h postdose [97]. In clinical trials, EVG was found to be well tolerated and efficacious [98]. Pharmacokinetic boosting with ritonavir (RTV) was found to result in improved dosing [99]. It is now known that the cytochrome p450 enzyme CYP3A4/5 is the primary metabolizing enzyme for EVG, followed by glucoronidation by UGT1A1/3 [99]. Thus, the bioavailability and clearance of EVG was found to be favored when EVG was dosed in combination with CYP3A4/ 5 inhibitors [74, 99, 100]. The CYP3A4/5 inhibitor, RTV, was found to cause an ~20-fold increase in area under the curve and to extend elimination half-life from 3 to 10 h [101]. In a phase II trial of ARV-naive patients (n048) starting initial therapy on an OBR of tenofovir/emtricitabine (TDF/FTC), coadministration of EVG with a novel pharmacokinetic booster, cobicistat, in a single tablet formulation resulted in undetectable viremia in 90 % of patients after 48 weeks, as compared with 83 % of patients who received TDF/FTC/EFV [102]. In a phase IIb study, RTV-boosted

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EVG was noninferior to the RTV-boosted PIs darunavir (DRV) and tipranavir (TPV), when other drugs were also employed in therapy [103]. However, despite being a once-daily drug, EVG shares a moderate genetic barrier to INI resistance with RAL, and extensive cross-resistance exists between the two compounds. The RAL signature mutations N155H, Q148H/R/ K, and G140A/C/S, as well as associated accessory mutations, were selected by EVG in culture [104] and in patients [93, 105]. This precludes the use of EVG to treat most RALresistant viruses. The only major RAL-associated mutations not selected by EVG were Y143C/R/H, and subsequent studies showed that viruses containing Y143C/R/H remained susceptible to EVG [106]. In addition to RALassociated resistance mutations, EVG selected for other mutational pathways. T66I did not confer high-level resistance to RAL [104] but conferred >10-fold resistance to EVG, while a T66R mutation conferred >10-fold resistance to RAL and >80-fold resistance to EVG [107, 108]. The T66I mutation is associated with a series of accessory mutations, including F121Y, S153Y, and R263K; the latter two have not been associated with RAL resistance [109]. A F121Y mutation has been selected with RAL and confers high-level resistance to this compound but has not yet been identified in the clinic [107]. Other clinically selected EVG mutations are S147G, which confers >8-fold resistance to EVG but does not affect susceptibility to RAL [107]. Other in vitro EVG selections resulted in several high-resistance mutations that have yet to be clinically validated, such as P145S, Q146P, and V151A/L [107]. The V151L mutation confers≈8-fold cross-resistance to RAL and has been identified in a single patient treated with RAL [110].

Second-Generation Integrase Inhibitors MK-2048 Efforts have been made to produce second-generation STIs with activity against RAL-resistant viruses. Optimization of tricyclic 10-hydroxy-7, 8-dihydropyrazinopyrrolopyrazine1, 9-dione compounds led to the development of MK-2048 [111] (Fig. 1) that demonstrated an EC95 330 and >140, respectively. In vitro combination antiviral studies showed that DTG did not increase toxicity when used in combination but had a synergistic effect with each of EFV, NVP, stavudine, abacavir, LPV, amprenavir, and enfurvitide, as well as an additive effect in combination with maraviroc. The HBV drug adefovir and the HCV drug ribavirin had no effect on the efficacy of DTG [125], allowing for its potential use in treating co-infected individuals. From a pharmacokinetic perspective, the profile of DTG allows once-daily dosing without pharmacokinetic boosting. This is based on a long unboosted half-life (13–15 h) with trough levels of DTG being much higher than the in vitro IC90 [126]. The side effects of DTG in HIV-infected volunteers were similar to those of placebo in phase I clinical trials [126]. Phase IIa randomized double-blind trials provided vital evidence of the anti-HIV effect and potency of DTG [127, 128]. Notably, 35 ARV-experienced INI-naïve patients, who were not receiving therapy and whose plasma HIV-1 RNA levels ranged from 3.85 to 5.54 log copies/mL, received once-daily doses of 2, 10, or 50 mg of DTG or placebo for 10 days. More than 90 % of patients who received DTG, irrespective of dose, had a decline in viral load to 200 cells/mm3 and HIV-1 RNA>1,000 copies/ mL, who were treated once daily with DTG (n0155) at 10-, 25-, or 50-mg doses or 600-mg EFV (n050) combined with background therapy of TDF/FTC or abacavir/3TC [129]. Over 90 % of all participants in the DTG arm had undetectable viremia after 24 weeks of treatment, establishing the noninferiority of DTG to EFV in an NRTI/NNRTI background and also showing that DTG was at least as safe as EFV. However, no primary INI resistance mutations have yet been reported for DTG either in culture or in the clinic. Tissue culture selection studies over 112 weeks identified, in order of appearance, viruses harboring T124S/S153F, T124A/S153Y, L101I/T124A/S153F, and S153Y by week 84. These mutations persisted throughout serial passaging but did not confer high-level resistance to DTG [125]. Position 124 of IN is modestly polymorphic, and S153F/Y had previously been described in EVG selection studies [130]. Despite an apparently high genetic barrier for resistance, selection studies involving DTG are ongoing, and reports that a R263K mutation in IN may confer modest ≈3-fold resistance to DTG have been presented [131]. DTG may enjoy a high barrier for resistance due to a higher affinity of DTG for IN, as compared with RAL and EVG [132]. This hypothesis has been previously suggested for MK-2048, which also has a relatively high barrier for resistance, since it also has high affinity (t1/2 032 h) for IN, as compared with RAL (t1/2 ≥7.3 h) [133]. Biochemical assays also showed that DTG exhibited tighter binding and had a longer dissociative half-life from IN than did either EVG or RAL [133]. An inverse relationship existed between the half-life of binding and the inhibitory potential of INIs when the binding half-life t1/2 was below 4 h. An FC in regard to drug resistance >3, relative to wild type, was observed when the t1/2 dropped below 1 h [132]. In these assays, the t1/2 of DTG, RAL, and EVG for wild-type enzyme were 71, 8.8, and 2.7 h, respectively. The fact that RAL and EVG have a shorter t1/2 than does DTG suggests that resistance mutations that affect binding of RAL and EVG might also be more likely to compromise antiviral potency. The notion of a relationship between dissociative t1/2 and antiviral potency is further supported by data on the E92Q/N155H, E138K/ Q148R, and G140S/Q148R substitutions [132]. DTG in INI salvage therapy is being investigated in an ongoing study termed VIKING. This is a phase II singlearm study investigating the feasibility of replacing RAL

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with DTG in patients experiencing failure due to RALresistant viruses [119]. Participants (n027) were switched from their previous RAL-containing regimens to receive 50 mg once daily DTG for 10 days and were then prescribed other active drugs over a period of 23 weeks. Eighteen of the study participants had INI-resistant viruses belonging to the Y143, Q148, and N155 pathways prior to initiation of the study. After 10 days of DTG monotherapy, all participants harboring viruses in the Y143 and N155 pathways attained a mean HIV-1 RNA decrease of ≈1.8 log copies/mL, as compared with ≈0.7 log copies/mL for viruses harboring G140S/ Q148HKR double mutations. None of the viruses harboring Q148HRK plus two or more additional mutations experienced a decrease of ≥0.7 log copies/mL, indicating a degree of resistance on the part of Q148HKR viruses to DTG. This trial nonetheless provided proof-of-principle for the use of DTG in RAL-experienced patients infected by subtype-B viruses harboring position Y143 and N155 substitutions. To model the effects of DTG in RAL-experienced patients, several serial passaging studies have been carried out and have shown that the presence of the N155H and Y143CHR resistance did not lead to development of additional resistance mutations under DTG pressure or to a substantial decrease in DTG susceptibility [61, 63]. In contrast, the presence of Q148HKR mutations did lead to further mutations and >100 FC for DTG susceptibility relative to wild type in subtype B viruses [123, 125]. Interestingly, Q148HKR mutations did not affect susceptibility to DTG in HIV-1 subtype C and HIV-2 isolates [125, 134, 135]. An ongoing trial termed SPRING-2 will evaluate the use of once-daily DTG versus twice-daily RAL in treatment-naïve patients. A phase III trial termed SAILING will compare once-daily versus twice-daily DTG in ARV-experienced INI-naïve HIV-positive subjects [136]. S/GSK-1265744 S/GSK-1265744 is a backup drug to DTG that has been tested in double-blind randomized placebo-controlled trials (Fig. 1) and has shown promising short-term efficacy, an excellent pharmacokinetic profile, and good tolerability in HIVinfected patients [137]. Although its future development is uncertain, given the positive state of development and promise of DTG, its long half-life and potential for nanoformulation may give it potential for use in HIV prevention studies.

Integrase Inhibitor Discovery Quantitative Structure–Property/Activity Relationships (QSPR/QSAR) The recent elucidation of the full-length prototype foamy virus (PFV) structure [138], intasome [139], and strand

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transfer complexes (STCs) [140–142] with DNA ligands and, subsequently, with RAL, DTG, EVG, and MK-2048 [139, 140] has allowed for the generation of homology models of HIV-IN that can be used to “train” and score drug prototypes. There are multiple QSPR/QSAR protocols and programs, some of which require advanced programming and mathematical skills, but several stand-alone and online programs offer semiautomated drug docking and scoring capabilities with moderate to high accuracy. The main aim of these approaches is to allow in silico validation and testing of prototype molecules in order to lower the costs associated with large-scale synthesis of nonvalidated compounds [143]. A recent summary of computer-based approaches for design of novel INIs that target 3′ processing, IN multimerization, strand transfer complex assembly, and IN–host-protein interactions has been published [144]. However, it is difficult to accurately model drug toxicity, bioavailability, and safety prior to the synthesis and study of novel potentially useful compounds.

Newer Strand Transfer Inhibitors in Preclinical Development MK-0536 by Merck & Co was designed on the basis of quantitative structure–property/activity relationships (QSPR/QSAR) that took into account the optimum minimum structure necessary for activity and generated a set of potential structures that could be synthesized and screened. MK-0536 has shown low hepatocyte clearance values [145] and generally good inhibition of wild-type IN and RAL-resistant IN [145, 146], but its current level of clinical development is unclear. Other classes of compounds that block strand transfer with high specificity at subnanomolar EC50s and low toxicities are cathechol-based [147], pyrimidone-based [148–151], dihydroxypyridopyrazine-1,6-diones [152], and quinolones, among others [153, 154].

IN–Host Factor Interactions IN takes part in a number of interactions with host proteins, and their posttranslational modifications have led to the targeting of some of these processes [155, 156]. Separate studies have shown that sumoylation [157] and acetylation [158] of IN occurs in vivo, leading to increased activity, but there are currently no specific inhibitors of these reactions that can specifically target IN modification without affecting other cellular proteins [159]. The observation that integration can be inhibited in ex vivo HIV-infected CD4+ T-cells of elite controllers [159] has not yet led to the identification of a responsible cellular factor. Currently, the most

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promising inhibitors targeting IN–host interactions disrupt interaction between IN and LEDGF/p75; the latter is a host protein that has been shown to be essential for tethering the IN preintegration complex (PIC) to host chromatin and also for the recruitment of other cellular factors to the PIC, thereby facilitating effective integration [160, 161]. Integration cannot occur in the absence of LEDGF/p75, and inhibition of the LEDGF/p75-IN interaction can block viral replication [160, 162]. This is supported by the recent finding that polymorphisms in the PSIP-1 gene that codes for LEDGF/p75 can affect rates of HIV disease advancement [163].

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specific yet dual-targeting inhibitors of both enzymes. Several early compounds that have been found to target both enzymes are DKAs [169, 170]. This hybrid class has been reviewed [171].

Integrase Inhibitors and HIV Diversity

LEDGINS were designed as specific small molecular inhibitors of the LEDGF/p75 interaction. 2-(quinolin-3yl) acetic acid derivatives have been co-crystallized with LEDGF/p75-IN, and optimized structures within this group were shown to inhibit the LEDGF/p75-IN interaction at sub-μM concentrations [160, 162]. Peptides mimicking the IN-binding domain of LEDGF/p75 exhibit potent inhibition of IN [164, 165]. A number of recent in-depth reviews have appeared on the topic of LEDGF/p75 targeted INIs [162, 165, 166].

Subtype differences may exist in regard to development of resistance to IN inhibitors, a phenomenon that also exists with RT inhibitors [87, 172, 173]. Despite the fact that HIV-1 subtype B and C wild-type IN enzymes are similarly susceptible to clinically validated INIs [121], the presence of resistance mutations may differentially affect susceptibility to specific INSTIs [87]. Recent reports suggest that the G118R mutation, which was previously reported to confer slight resistance to MK-2048, imparts a 25-fold resistance to RAL when present together with the polymorphic mutation L74M in CRF-AG cloned patient isolates [174]. Additionally, it is well documented that the INI Q148RHK resistance mutations, which affect susceptibility to DTG in HIV-1 subtype B, may not affect the susceptibility of either HIV1 subtype C or HIV-2 enzymes to DTG and other INSTIs [132].

3′ Processing Inhibitors

Major Messages

BI 224436

–

Blocking of LEDGF/p75-IN Interaction

This compound is a novel INI with a distinct mode of action from more established INSTIs. It is a noncatalytic site INI (NCINI) (Fig. 1) that interferes with the interaction between IN and the chromatin targeting the LEDGF/p75 protein, yielding low nM inhibition of 3′ processing and viruses [167]. The profile of this compound appears favorable, and it also appears to be specific, since it did not exhibit reduced activity against any INSTI-resistant IN enzymes [167]. This compound has now entered phase Ia clinical trials to evaluate dosing and safety in healthy individuals. Initial reports indicate high bioavailability with good tolerability at single doses ranging up to 200 mg. BI 224436 also exhibited good dose-proportional pharmacokinetics when given as a single dose of 100 mg, and plasma levels appeared adequate to achieve benefit [168].

– –

–

New INI resistance mutations are G118R, R263K, and S153Y, some of which confer cross-resistance among second-generation INSTIs. Structural modeling and deep sequencing have contributed to a better understanding of the role of INSTI resistance mutations. INSTI resistance is dynamic and can lead to the selection of mutations at position Q148 that are associated with broad cross-resistance to all members of this class to an extent that far exceeds that associated with the Y143 and N155 resistance pathways. Several new INIs that include novel INSTIs, as well as compounds that target other IN functions, are currently being developed.

Acknowledgments We thank the Canadian Institutes of Health Research (CIHR) and the Canadian Foundation for AIDS Research (CANFAR) and ISTP Canada for support. P.K.Q is a recipient of a CAHR/CIHR Doctoral Scholarship. T.M is the recipient of the BMS/ CTN Postdoctoral Fellowship.

Dual-Acting RT/IN Inhibitors Structural and functional similarities between HIV-1 IN and the RNAse-H domain of HIV-1 RT suggest the possibility of

Disclosure No potential conflicts of interest relevant to this article were reported.

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