The development of resistance of HIV-1 to zalcitabine (1997)

Introduction Antiviral therapies against HIV infection are known to select for variants with reduced sensitivity (resistance) to the antiviral agent. The development of high-level viral resistance has been temporally associated with the loss of favourable effects on activity markers (e.g., CD4+ cell count and plasma viral RNA levels) with a number of antiretroviral agents. For example, the emergence of resistance mediated through the Met184Leu mutation during lamivudine (3TC, 2′3′-dideoxy-3′-thiacytidine) therapy is found to correlate with at least partial loss of antiviral effect of this agent [1–3]. Selection of resistant virus, therefore, represents a major cause of failure of antiviral therapy. Furthermore, a relationship between the development of resistance to zidovudine (ZDV, 3′-azido-2′3′-dideoxythymidine) and increased risk of clinical progression or death has been reported, raising concerns that appearance of resistant virus may have negative consequences for patients [4–7]. Resistance to ZDV has been reported in 10–15% of persons presenting with primary HIV-1 infection in urban areas [8,9], underlining the importance of starting antiretroviral therapy with more than one agent. Rationales for combination therapy also include (i) the potential for antiviral synergy, as has been reported in vitro with a range of agents both from the same class, for example, ZDV and zalcitabine (ddC, 2′3′-dideoxycytidine) [10], and different classes, for example, ZDV or ddC or both with proteinase inhibitor [11]; and (ii) the potential to delay or prevent resistance to one or more agents in the combination. The benefits of combination therapy on disease progression and survival have been reported recently with ZDV-ddC or ZDV-didanosine (ddI, 2′3′-dideoxyinosine) therapy compared with ZDV monotherapy as initial therapy [12–14], and ddC-saquinavir (SQV) compared with either agent alone in ZDV-experienced patients [15]. There remains an urgent need not only for new antiviral drugs, but also for new regimens of drug combinations. Despite a number of combination regimens achieving reductions in viral load over 1-year periods to below the level of detection of currently available assays, these regimens are likely to fail eventually in the majority of patients due the appearance of resistant virus, which arises from residual viral replication in less well penetrated ‘sanctuary sites’ (such as the central nervous system or genital tract) secondary to episodic compliance failure, or possibly from acquisition of drug-resistant virus from another individual. Long-term management of persons with HIV infection is therefore likely to involve the sequencing of combination regimens. Given the limited number of antiretroviral agents currently available, we must consider whether therapies can be optimally sequenced to attain the maximal magnitude and duration of reductions in viral load achievable with each regimen. This can only be possible by reserving those antiviral compounds which induce the greatest levels of resistance and cross-resistance for later use. Currently available antiretroviral therapies involve the inhibition of the viral enzymes reverse transcriptase (RT) or proteinase. The nucleoside analogue ZDV is the longest established and most widely used anti-HIV RT agent [16]. Other nucleoside analogue RT inhibitors include ddC, ddI, stavudine (d4T, 2′3′-didehydro-2′3′-dideoxythymidine) and 3TC. Two of these compounds, ddC and 3TC, share a common nucleotide base, but differ in their sugar moieties and stereochemistry (Fig. 1). Both are approved for use in combination with ZDV.Fig. 1: The structures of (a) zalcitabine and (b) lamivudine.This review summarizes some of the mechanisms of resistance and examines the resistance profile of ddC in vitro and in clinical trials. Genetic diversity and selection In common with many RNA viruses, HIV displays a high degree of genetic diversity, even within individual patients at any given time [17]. The very high rate of turnover of the virus [18,19] and the high rate of incorporation of errors by the HIV RT (an estimated in vivo forward mutation rate of 3.4×10−5) [20] are key to the continual generation of diversity. Indeed, it has been suggested that there may be ‘mutation-prone regions’ present in the HIV RT gene with higher rates of error incorporation [20,21] that are not (as with human DNA polymerases) ‘edited out’ because HIV RT does not possess detectable proof-reading exonuclease activity [22]. Such is the level of diversity that each infected individual is thought to harbour a very large, dynamic reservoir of virus quasispecies [23]. A large proportion of lymphoid CD4+ cells harbour HIV, mostly in latency [24]. However, it is estimated that of the order of 109 CD4+ lymphocytes are productively infected even in individuals with asymptomatic infection [25]. It is also noted that this approximates to the daily turnover of CD4+ lymphocytes in infected patients [18,19]. In addition, the half-life of plasma HIV-1 is thought to be of the order of 2 days or less and the daily turnover of virus is calculated to be at least 108−109 virions [18,19,26]. Together with the rate of error incorporation, this indicates a very high level of potential daily variability. One implication of this is the likelihood of mutations related to drug resistance being present in viral populations prior to therapy [27]. This makes the virus highly susceptible to positive selection. Under normal circumstances such selection is driven by the immune response. However, the dynamic response of the virus to external pressures such as vaccination or antiviral intervention provides a major barrier against the successful control of HIV-1 infection [28,29]. This is exemplified by the well-documented selection of HIV-1 variants resistant to antiviral agents in patients during therapy [30]. Selection pressure from the nonnucleoside RT inhibitor nevirapine can result in a complete replacement of wild-type with single-substitution highly resistant mutant within 14 days of therapy [19]. Other therapies require more prolonged treatment before resistant virus becomes predominant. For example, resistance to SQV is an infrequent event even after 1 year of therapy, particularly when combined with other agents, despite the requirement of only one or two mutations to establish resistance [31]. Resistance to other compounds such as ZDV, ritonavir and indinavir may require accumulation of several mutations before significant changes in median inhibitory concentration (IC50) are observed. However, the number of mutations required may not be predictive of ‘time to resistance’ in the clinic. Factors that may delay the appearance of drug-resistant virus include the following: (i) the extent of pre-treatment polymorphism at the critical mutation points; (ii) the influence of resistance mutations selected for by prior and concomitant agents; (iii) the effect of resistance mutations on viral replication competence; (iv) the selective pressure applied by a regimen; and (v) the extent of residual viral turnover during therapy. Rational therapy should, therefore, involve a very potent combination of antiretroviral drugs with non-overlapping resistance profiles that are known to significantly reduce viral load and delay resistance to fellow components of the regimen. In addition, the different profiles of resistance selected by antiviral agents in monotherapy and combination use should be carefully considered to limit narrowing of future therapeutic options. Structural basis for resistance to inhibitors of HIV RT HIV RT is a heterodimer composed of p66 and p51 subunits. The p51 subunit is a proteolytic cleavage product of p66. It comprises the amino-terminal 440 amino acids of p66. This region corresponds with the polymerase domain. The subdomains within this are designated fingers, palm, thumb and connection (Fig. 2) [32]. The additional carboxy-terminal 120 amino acids of p66 makes up an RNaseH domain.Fig. 2: The structure of the p66 subunit of HIV reverse transcriptase. The fingers, palm, thumb and connection domains are shown. In addition, the catalytic aspartic acid (D) residues and the main sites implicated in the generation of resistance to zalcitabine are indicated. Reprinted with the kind permission of Drs A. Kroehn and B. Sherborne (Roche Discovery Welwyn, Welwyn Garden City, Hertfordshire, UK).The RNA template-primer complex binds to a cleft between the subdomains of the polymerase region of p66 such that the 3′-OH terminus of the primer lies close to the polymerase active site. In p51, the binding cleft is absent, the essential aspartic acid residues of the active site are buried and the polymerase appears to be inactive. However, it should be noted that the mutations that give rise to reduced sensitivity to inhibitors of the polymerase function are likely to be present in both p66 and p51. The main features of the structure of the p66 subunit of HIV RT are shown in Fig. 2. Nucleoside analogues and dNTP bind at a site in the palm domain of p66, beside the 3′-terminus of the primer. This site includes the catalytic aspartic acids (Asp110, Asp185, Asp186) [33,34]. Gln151 is located close to the template strand and may be involved in base-pair recognition [35]. More recent data confirm that Gln151 is involved in dNTP binding and further implicate it in the nucleotidyl transferase catalytic step [36]. An essential role for this residue is suggested by the highly conserved nature of the sequence Leu-Pro-Gln-Gly (residues 149–152) among 26 different retroviruses including HIV, simian immunodeficiency virus and other animal retroviruses [37,38]. A conserved motif Ile63−Lys64−Lys65−Lys66, which is involved with both binding dNTP and the enzyme template-primer interaction [32,39], is also present. A monoclonal antibody targeted to amino acids 65–69 has been found to neutralize RT activity [39], suggesting an essential role for these residues. Resistance to ddC High-level (>100-fold changes in IC50) resistance to ddC has not been reported to date either from in vitro studies or from clinical trials. Furthermore, the development of resistance to both ddI and ddC seems to occur less frequently than to ZDV [40], possibly in part related to the greater similarity of these compounds to natural substrate compared with that of ZDV (or 3TC). Interpretation of changes in IC50in vitro should be made with consideration of the inherent variability of the assays (fourfold) [41]. In addition, because both ddC and ddI are most active in resting cells and assays generally use phytohaemagglutinin-stimulated lymphocytes, standard systems may not accurately reflect in vivo conditions [42]. Five individual mutations have been identified as being primarily involved in the generation of reduced sensitivity to ddC: Lys65Arg, Thr69Asp, Leu74Val, Gln151Met, Met184Val/Ile. It is notable that all these are situated within or close to conserved motifs near to the catalytic site. Lys65Arg Low-level resistance to ddC has been demonstrated following in vitro selection carried out with both MT4 cells and cord blood peripheral blood mononuclear cells (PBMC). A mutation (Lys65Arg) was found to confer 12-fold reduced sensitivity to ddC [43]. Reduced sensitivity (5–10-fold) was confirmed in a molecular clone and the mutation was identified in clinical specimens from four out of 11 patients treated with ddC for more than 2 months [43]. Reduced chain termination of HIV RNA transcription by RT was reported in Lys65Arg mutant RT in the presence of ddC, which was consistent with the increased IC50 measured in tissue culture with mutant virus generated by site-directed mutagenesis of a molecular clone [44]. Cross-resistance with dNTPs, 3TC-triphosphate (TP) and ZDV-TP was demonstrated in these studies by chain-termination measurements [44,45]. However, IC50 determinations in tissue culture indicated that although cross-resistance to ddC, ddI, 3TC and 9-(2-phosphonylmethoxyethyl) adenine (PMEA) could be demonstrated, reduction in sensitivity to ZDV was not observed [45–47]. This lack of correlation is not surprising since many factors may influence the sensitivity of HIV to compound in tissue culture. It has been reported that the choice of cell line may have a profound influence on results from this type of study [48]. In one study with recombinant mutant virus, cross-resistances of 10-fold against ddC, 20-fold against 3TC and threefold against ddI were reported [43]. Interestingly, a recombinant virus with two mutations (Lys65Arg and Met184Val) did not show increased levels of resistance to ddC over those found with the Lys65Arg mutant alone [43]. The a mutation to It was reported in a after treatment with ddC It appears to be to ddC and does not confer to ZDV or This is surprising since mutation of the amino acid sensitivity to ZDV also effect on sensitivity of virus to ddC The mutation a reduction in the ddC sensitivity of a molecular clone compared with the clone from a different therapy with ZDV and ddC for were found to have mutations at and and the mutations were in a molecular it was confirmed that the mutation a reduction of sensitivity to ddC, but it was also reported that the mutation at ddC sensitivity to ddC by a of reduced to Despite resistance to ddC was present in the clinical with mutations at both and other between these and the wild-type molecular clone have for this months of treatment with ddI monotherapy did not result in of the did resistance to ddI The mutation at has also been reported during ZDV-ddC combination therapy This mutation was in virus from an individual therapy with ddI It does not to a in vivo for ddC a molecular and reductions of sensitivity to ddI and ddC, were In combination therapy with ZDV, it should be noted that the of this mutant is very even after therapy one in one in In the is higher out of after 1 year of monotherapy with ddI In a study of the effects of it was reported that a virus from a molecular clone with the mutation was less to but did not show significant cross-resistance with the or This a role for the sugar in the of In a recent virus was from a ZDV-ddC for ZDV and ddC resistance was observed that this a of not reported with either ZDV or In were of which are thought to to The mutation was in of essential role in the RT enzyme The mutant virus a in ZDV in ddC and in ddI of from this have demonstrated that the mutation at which there was a concomitant in This was by and and and which increased with these mutations has been more reported in persons therapy mutagenesis studies with the virus that the mutation a reduction in sensitivity to ddC, a 10-fold reduction to ZDV and a reduction to ddI The mutant also reduced sensitivity to and to 3TC However, although the of the four further mutations in a molecular clone increased the level of resistance to ZDV and ddI to and resistance to ddC was further increased by only a of two In other mutations were to the molecular clone this genetic all mutations only a 10-fold reduction in sensitivity to ddC Interestingly, the mutation at was found to confer reduced although this was for by the mutation at of from two the mutation at was found to that at The by of the of a mutation at before that at that such a remains in the In a in vitro study with of virus in the presence of ddI, a with cross-resistance to ddC was and found to have the mutation recombinant virus, it was found to a reduction of sensitivity to both ddI and ddC, but with effect on ZDV The mutation was reported in out of patients ddI therapy combined with ZDV studies have suggested a one out of patients treated with for and two out of in a ZDV-ddC for the same The mutation was observed after of HIV-1 in and cells in the presence of and The of HIV-1 variants highly resistant to 3TC and was found after only two to cell High-level resistance of both variants for the and of both compounds was to ZDV was but a reduction in sensitivity to ddC was observed Two and were The was confirmed in studies with and recombinant virus, although in this the observed resistance to ddC was of the order of only threefold This mutation is to two of the catalytic aspartic acid residues and may influence enzyme but does not to RT activity In the mutation seems to more than the mutation This may be to the higher of the DNA for compared with the for However, over wild-type virus, is in with This may the mutation is most observed during both and combination therapy with 3TC, although it may be by the appearance of the mutations have been shown to occur and at high in patients treated with 3TC monotherapy or in combination therapy. Resistance has been within 1 of and increased in to of patients within A reduction of sensitivity of the order of to to 3TC with associated reductions of sensitivity to ddC to and ddI to are in clinical This level of resistance to 3TC is associated with at least partial loss of antiviral but not of viral load to The partial of antiviral effect despite high-level resistance to 3TC has been suggested to be related to the influence of mutations at on viral in as be from It has been noted in vitro that the mutation on the genetic of the resistant at least ZDV sensitivity on virus reduced to An in sensitivity of virus (e.g., to to has also been found in some persons after 3TC treatment in the In addition, although combination treatment results in the acquisition of there is some that it a delay in the appearance of ZDV resistance However, there is both in vitro and from clinical that RT may to the and virus with resistance it is whether additional mutations are required for data of the RT connection or In a clinical study of patients in which 3TC was combination with ZDV after at least months prior therapy with this after resistance to 3TC. patients with ZDV resistance at 3TC four virus of the resistance The of selection for these mutations at during 3TC therapy has concerns that prior therapy with this agent may limit future therapeutic from ddC or ddI A clinical is to this of resistance to ddC and possible clinical it from the that there are to the virus to resistance to ddC in it is that these are up by the virus during combination therapy. ddC resistance from monotherapy studies have been In studies have ddC resistance during ZDV-ddC therapy. In study in to ddC after therapy was observed in although two out of from patients treated with resistance to ddC or ddI were noted to influence the development of resistance to ZDV in the study ddC resistance was not found in 11 from patients treated for up to or in patients treated for up to Furthermore, in the from the a of patients after therapy with ZDV ZDV-ddC or were found not to have any mutations associated with resistance to ddI or ddC In a study of ZDV-experienced from patients of ZDV-ddC therapy, Lys65Arg and mutations can give cross-resistance to were not although were at particularly as initial therapy, provides significant delay in clinical disease progression compared with ZDV monotherapy and in viral load and over to those observed with and The benefits of this combination may from both with ZDV against both and [10], and viral sensitivity to of this combination with SQV the development of SQV resistance in vivo to SQV monotherapy [31]. SQV has been noted to delay ZDV resistance in vivo therefore, the combination of currently used in clinical includes agents that delay resistance to each of the agents, a that may be in the more and effects on activity markers observed with this combination compared with The influence of ddC resistance on clinical or activity has not been The presence of ZDV resistance is associated with increased risk of disease However, of viral sensitivity is only one in therapy, in drug of other such as cell the presence of a viral and viral load and markers of both risk of future progression and therapeutic response. Furthermore, because treatment sequencing have been reported, the extent of influence of changes in in vitro sensitivity to a compound on clinical activity of agents is of viral sensitivity to an antiviral drug provides on other of viral that may be by mutations in RT such as replication enzyme and A number of mutations that resistance to ddC have been However, the of their in combination with the levels of sensitivity changes observed and the limited cross-resistance of ddC therapy. Indeed, it that from ddC therapy is more at risk of being by the selection of virus through the use of ddI or 3TC than the use of ddC is the of mutations to induce high-level resistance It has been suggested that the sequence for nucleoside analogue therapy ddC of ddI on the basis of both activity in and patients and cross-resistance The being that cross-resistance selected by ddI at is likely to reduce the of ddC therapy. it be that use of 3TC should be for use after ddI as of cross-resistance to both ddC and ddI the potential of these two in a high proportion of patients most have not noted cross-resistance between ZDV and ddC, in from ddC and patients treated with ZDV, a significant correlation was found between the development of resistance to ZDV and reduced sensitivity to ddC and ddI The molecular basis of these remains but it is that the antiviral activity of ZDV-ddC therapy is greater in patients not resistance to ZDV A combination of two nucleoside analogues with a agent from a different represents the standard of antiretroviral therapy in most The combination of ZDV-ddC appears to clinical in patients to and effects on activity markers to it is therefore to between these agents on the basis of The of in studies with at a of disease is [12–14], and the only study with a study to of ZDV-experienced persons with cell found in the of these that the ZDV-ddC combination in patients with cell represents an for therapy. Other nucleoside which may in ZDV-experienced patients and possess greater potential for virus, could be considered as options. of antiviral effect of the ZDV-ddC (or combination appears to be associated with resistance to ZDV but not it to the ZDV of the for an agent of non-overlapping resistance profile However, as more agents available most may to both of the regimen. In ddC appears used in combination with SQV (or possibly other proteinase as this combination provides a survival over ddC monotherapy and ddC may delay resistance to SQV [31]. Such is the dynamic nature of HIV that the of virus be to in their to the main anti-HIV antiviral agents. Indeed, in of the of ZDV resistance in which appears to be there is a for therapy with combinations. ddC in combination with SQV represents an with clinical and a potential for cross-resistance to both other RT and proteinase inhibitors In of resistance and cross-resistance should be considered when antiretroviral treatment with the of achieving maximal therapeutic the possible of future therapeutic options. to be by the combination of ZDV and on the of resistance during therapy with drug and of mutant and the influence of drug resistance on disease progression and of future agents is required to therapy