Chapter 4Resistance to entry inhibitors

Poveda E, Soriano V.

Publication Details

Introduction

Viral entry is currently one of the most important targets in the search for new drugs to treat HIV-1 infection. Advances in the knowledge of the molecular mechanisms involved in the different stages of the entry process have allowed the production of molecules that block each step: (1) attachment of the viral glycoprotein gp120 to the CD4 cell receptor; (2) binding of the gp120 to chemokine co-receptors CCR5 or CXCR4; and (3) the gp41-mediated fusion of the viral and cellular membranes. Entry inhibitors are the latest family of antiretroviral compounds to come into clinical use. The first compound to be approved was the fusion inhibitor enfuvirtide [13]. Many other entry inhibitors are currently in clinical development and it is hoped that they will soon be part of the therapeutic armamentarium against HIV-1. This new family of antiretroviral agents is eagerly awaited by the growing number of patients carrying drug-resistant viruses to reverse transcriptase and protease inhibitors. However, clinical experience has taught us that HIV-1 almost always finds a way to escape, mutating and evading drug-selective pressure [4]. In this chapter, in vitro and clinical data concerning the mechanisms of resistance to entry inhibitors will be reviewed (Table 1).

Table 1. HIV entry inhibitors: mechanisms of action and possible resistance pathways.

Table 1

HIV entry inhibitors: mechanisms of action and possible resistance pathways.

Resistance to CD4–gp120 binding inhibitors

The first step in the viral entry process is the attachment of the viral gp120 to the CD4 receptor present in the cell surface. It is mainly driven by electrostatic forces between the positive charge of the CD4 molecule and the negative charge of the gp120 pocket. Van der Waals' forces and hydrogen bonds help to stabilise the initial CD4–gp120 interaction. It is estimated that the amino acid Phe 43 in the CD4 receptor accounts for 23% of the binding affinity to HIV-1 gp120 [5]. Following CD4–gp120 binding, gp120 undergoes conformational changes that allow the subsequent interaction with CCR5 or CXCR4 on the cell surface.

There are many candidate inhibitors of CD4–gp120 binding, including PRO-542 [CD4-immunoglobulin G2 (IgG2)], TNX-355, BMS-806 and CADA, each characterised by a different structure and mechanism of action [68]. Information on resistance to these inhibitors is scarce. In vitro studies of BMS-806 and related compounds (e.g. BMS-155) have shown that the amino acids of gp120 implicated in resistance are those surrounding the Phe 43 pocket. Changes in five gp120 residues (Trp 112, Thr 257, Ser 375, Phe 382 and Met 426) result in escape from inhibition by BMS-806 and BMS-155. The degree of sequence conservation of the nearby V1/V2 variable loops indirectly influences the sensitivity to these drugs [9]. Natural gp120 variability among different HIV-1 subtypes may account for differences in baseline susceptibility to this class of compounds. This is the case for subtype C and recombinant subtype AE (CRF01_AE), which seem to be naturally resistant to BMS-806 [10].

Resistance to CCR5 and CXCR4 antagonists

In the second step of viral entry, the CD4–gp120 interaction provokes conformational changes in the viral envelope that allow the CD4–gp120 complex to interact with the CCR5 or CXCR4 chemokine co-receptor. The V3 loop in HIV-1 gp120 is the main domain involved in this interaction and V3 amino acid sequences largely determine the preferential use of CCR5 or CXCR4 by HIV-1 as a co-receptor for entry into the cells [11]. Accordingly, HIV-1 isolates may be classified as CCR5 tropic (R5), CXCR4 tropic (X4) or dual tropic R5/X4 strains, depending on their co-receptor use [12].

Several compounds designed to block the CCR5–gp120 interaction of R5 strains are currently in clinical development, including SCH-C, vicriviroc (SHC-D), aplaviroc (GW873140), maraviroc (UK-427,857), TAK-220, TAK-652 and PRO-140. The sites of interaction of these molecules with CCR5 have been mapped within the pocket formed by the transmembrane helices of CCR5 [13]. The binding of CCR5 antagonists with the co-receptor disrupts the conformation of CCR5, specifically the second extracellular loop (ECL2), which can no longer interact with the V3 loop of HIV-1 gp120 [14,15]. Some inhibitors, such as aplaviroc, appear to interact directly with ECL2 [16]. The clinical development of this compound, however, has recently been halted (at least in drug-naive patients), due to the development of liver toxicity in some patients [17].

Two main resistance pathways have been described for HIV-1 to escape from CCR5 or CXCR4 antagonists. The first is a shift in co-receptor usage. The second results from changes in regions of the viral envelope that allow the interaction between gp120 and the co-receptor to occur despite the presence of the inhibitor. An increased affinity of gp120 for CCR5 has been shown to enable more efficient competition of HIV-1 with CCR5 inhibitors for binding to the CCR5 co-receptor [18]. Data available to date suggest that most CCR5 antagonist-resistant strains preserve the use of the CCR5 co-receptor, and that multiple mutations within different regions of gp120 [V3, V2 and constant regions 2 and 4 (C2 and C4)] account for the drug-resistant phenotype [1921]. It should be noted that most mutations are specific for each compound, which may limit cross-resistance and allow sequencing within this class of compounds. However, large clinical studies are needed to confirm these in vitro observations. Preliminary findings with viral isolates resistant to maraviroc have demonstrated that they remain susceptible to SCH-C, vicriviroc and aplaviroc [22]. In contrast, vicriviroc-resistant strains show cross-resistance to SCH-C, AD101 and regulation on activation, normal T cell expressed and secreted (RANTES) derivatives, most probably because they share the site of interaction with CCR5 [21]. It is clear that CCR5 antagonist-resistant strains do not show cross-resistance to the currently approved reverse transcriptase and protease inhibitors. In addition, there is no evidence of cross-resistance with other classes of entry inhibitors, including CD4–gp120 binding inhibitors and enfuvirtide [21].

CXCR4 antagonists block the interaction of the CD4–gp120 complex with the ECL2 domain of the chemokine co-receptor CXCR4 [2325]. Some of these compounds, such as AMD-070, KRH-1636 and KRH-2731, are currently in pre-clinical and clinical development [8], but information on selection of resistance is scarce. Mutations in the V3 domain seem to account for the loss of susceptibility to many of these drugs. However, changes in other gp120 regions (V1, V2 and V4) have also been observed, including deletion of five amino acids (364–368) within the V4 domain [26,27].

Preliminary results have failed to identify a shift in co-receptor use as the main resistance pathway for evading CXCR4 antagonists; however, it will be necessary to wait for the results of ongoing larger clinical trials to confirm this. It should be noted that while the demonstration of a shift in co-receptor use from CCR5 towards CXCR4 could accelerate HIV-1 disease progression, the contrary could be the case using CXCR4 antagonists, given that R5 viruses tend to be associated with less virulent HIV-1 disease. It is worth noting that recent data suggest that the consideration of a patient's virus population as R5 or X4 is too simple. Most subjects carry a predominant virus population with the other present as minor quasispecies. Under treatment with CCR5 antagonists, replacement of R5 by X4 viruses could occur as the result of drug pressure. In most cases, the virus population emerging as predominant was present already, although it represented a low proportion of the total virus population.

Resistance to fusion inhibitors

Following the interaction between viral gp120 and the cell CD4 receptor, and the interaction with chemokine co-receptors, another conformational change occurs in the viral envelope that causes a shift from a non-fusogenic to a fusogenic state. The viral gp41 protein, which comprises the repeat regions 1 (HR1) and 2 (HR2), drives the fusion process. The N-terminus domain of gp41 is exposed and inserted through the fusion peptide (FP) into the cellular membrane, allowing viral and cellular membrane fusion. Thereafter, the viral capsid enters the cytoplasm [3,28].

Enfuvirtide, the first entry inhibitor to be approved for clinical use, is a synthetic peptide that mimics the HR2 fragment of gp41. Its binding to the HR1 region blocks the formation of a six-helix bundle structure, which is crucial for the fusion process. Early in vitro studies showed that resistance to enfuvirtide involved the selection of changes in three amino acids (positions 36–38) within the HR1 region of gp41 [29]. However, further research has revealed that virological failure in patients receiving enfuvirtide therapy may involve the selection of changes in a larger fragment of HR1, from codons 36 to 45 [3032] (Figure 1). As expected, the impact on the loss of susceptibility to enfuvirtide caused by changes at different positions varies considerably, although it appears that changes in simple amino acids may be responsible for high-level resistance to enfuvirtide in some cases [33]. Therefore, enfuvirtide should be considered as a drug with low genetic barrier for resistance. The clinical implication of this fact is that the optimal use of the drug is in combination with other active antiretroviral compounds and never as the only active agent in a regimen.

Figure 1. The gp41 lineal structure and enfuvirtide sequences that mimic HR2.

Figure 1

The gp41 lineal structure and enfuvirtide sequences that mimic HR2. FP, fusion peptide; CC, cysteine–cysteine; tm, transmembrane domain; NH, N-terminal.

There is a wide range of susceptibility to enfuvirtide in viral isolates collected from enfuvirtide-naive patients, as well as from individuals undergoing enfuvirtide therapy and harbouring the same resistance mutations [31,33,34]. The determinants of this broad heterogeneity in virus susceptibility are unclear, but naturally occurring polymorphisms in the HR2 region of gp41 as well as changes in HR2 selected during therapy with enfuvirtide may explain this phenomenon [35]. Changes in HR2 have been observed in patients receiving long-term enfuvirtide therapy, although they do not seem to follow a consistent pattern. Therefore, at present, it is difficult to conclude how changes in HR2 influence susceptibility to enfuvirtide [33,36]. In addition to being directly implicated in enhancing resistance, changes in HR2 may also act as compensatory mutations, thus improving viral fitness [37].

Controversy exists regarding the impact of HIV-1 co-receptor tropism on the susceptibility to enfuvirtide. Although some in vitro studies have shown that R5 strains are resistant to enfuvirtide [38,39], in vivo studies have not found significant differences in response to enfuvirtide therapy when comparing patients harbouring R5 or X4 strains [40,41].

In addition to viral factors, host determinants may also influence the susceptibility to enfuvirtide. A relationship between the level of co-receptor expression on target cells and fusion kinetics has been found, such that the presence of high levels of CCR5 on the cellular surface results in more rapid membrane fusion, reducing the time in which gp41 could be targeted by enfuvirtide. Thus, individuals carrying delta-32 deletions in the CCR5 co-receptor (Δ32-CCR5), who express low levels of CCR5, might consequently respond more favourably to enfuvirtide [39].

Conclusions

Entry inhibitors mark the beginning of a new era in the history of antiretroviral therapy, opening new therapeutic options for the already large and growing number of patients carrying drug-resistant viruses. Enfuvirtide is the first agent of this class approved for clinical use. Several other compounds are currently in clinical development and may soon be available for use in the treatment of HIV-1. Available evidence indicates that selection of drug resistance may occur with these compounds. However, the pathways leading to resistance to entry inhibitors differ substantially from those causing resistance to the antiretrovirals in current use, and therefore no cross-resistance is anticipated between entry inhibitors and other classes of antiretrovirals, thus allowing salvage therapy with entry inhibitors.

The main mechanism of resistance to enfuvirtide is the selection of changes in a domain consisting of 10 amino acids, between residues 36 and 45 in the HR1 region of gp41. For other entry inhibitors, multiple changes in different gp120 domains (V3, C2, C4 and V4) seem to be responsible for causing loss of susceptibility, although with limited cross-resistance in most cases. Finally, natural susceptibility of different HIV-1 variants to entry inhibitors warrants further investigation, given that most entry inhibitors target the most variable HIV-1 proteins.

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