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Resistance to nucleotide analogue inhibitors of hepatitis C virus NS5B: mechanisms and clinical relevance
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ScienceDirect Resistance to nucleotide analogue inhibitors of hepatitis C virus NS5B: mechanisms and clinical relevance Matthias Go¨tte1,2,3 The high barrier to the development of resistance to nucleotide analogue inhibitors of the hepatitis C virus (HCV) RNAdependent RNA polymerase is an intriguing property of this class of drugs. The S282T substitution in the viral polymerase confers resistance to 20 -C-methylated nucleotide analogues. Although this mutation can be selected in HCV replicons, it has only been identified in very few cases in the clinic. Alternative resistance pathways are likewise rarely seen in vivo. Possible underlying mechanisms that are associated with the selection and establishment of a resistant genotype are discussed in this review. Addresses 1 Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec H3A 2B4, Canada 2 Department of Biochemistry, McGill University, 3655 Sir William Osler Promenade, Montreal, Quebec H3G 1Y6, Canada 3 Department of Medicine, Division of Experimental Medicine, McGill University, 1110 Pine Avenue West, Montreal, Quebec H3A 1A3, Canada Corresponding author: Go¨tte, Matthias ([email protected])
Current Opinion in Virology 2014, 8:104–108 This review comes from a themed issue on Antivirals and resistance
(HCV) RNA-dependent RNA polymerase or non-structural protein 5B (NS5B).
Development of nucleotide analogue NS5B inhibitors In 2003, Carroll and colleagues reported that the triphosphate form of 20 -C-methylated and 20 -O-methylated nucleoside analogues are efficient inhibitors of HCV NS5B [1]. These compounds also showed antiviral activity in subgenomic replicon cells. 20 -C-methyladenosine was more potent than 20 -O-methylcytidine, which has been ascribed to higher intracellular concentrations of 20 C-methyladenosine triphosphate. Subsequently, Migliaccio and colleagues reported the selection of resistant replicons with a single S282T mutation HCV NS5B [2]. The isolated mutant variant also conferred decreased susceptibility to 20 -C-methylguanosine. Another chemical class of nucleotide analogues that contain the 40 -azido modification select for the S96T substitution in NS5B [3]. Cross-resistance between the two different classes of NS5B inhibitors is commonly not observed. Both 20 -methyl-modified and 40 -azido-modified compounds show generally pan-genotypic activity.
Edited by Luis Mene´ndez-Arias and Douglas D Richman
http://dx.doi.org/10.1016/j.coviro.2014.07.010 1879-6257/Published by Elsevier B.V.
Various prodrug approaches were developed to increase bioavailability and to address limitations associated with the intracellular conversion to the active triphosphate form [4,5]. NM283, a prodrug of 20 -C-methylcytidine, was the first nucleotide analogue under clinical investigation for treatment of HCV infection. However, toxicities and/or limited potency caused the discontinuation of several of these first-generation nucleotide analogues, including NM283 and R1626, that is, the prodrug of 40 -Cazidocytidine.
Introduction Nucleotide analogue inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) remain important components in currently available treatment regimens. The diverse and often complex resistance patterns associated with the use of these drugs have been extensively described. Alternative genetic pathways that can involve either single or multiple amino acid substitutions in HIV-1 RT also provide various defined biochemical mechanisms. These previous findings raised the question whether similar diverse and complex resistance patterns may also evolve in the context of novel nucleotide-based therapies against chronic hepatitis C infection. The purpose of this review is to discuss nature, mechanistic aspects, and relevance of resistance to nucleotide analogue inhibitors of the hepatitis C virus Current Opinion in Virology 2014, 8:104–108
Next generations of nucleotide analogues include mericitabine and sofosbuvir. Mericitabine is currently evaluated in advanced clinical trials [6], while sofosbuvir is already approved for the treatment of chronic hepatitis C infection [7]. Both drugs share the 20 -a-F-b-C-methyl motif of the sugar, and differ in the nature of their base moiety and prodrug approach. Mericitabine is a 20 -a-F-bC-methylcytidine with a hydrolysable 30 , 50 -diester moiety. Sofosbuvir is a 20 -a-F-b-C-methyluridine with a phosphoramidate that, following hydrolysis, yields the 50 -monophosphate of 20 -a-F-b-C-methyluridine. This prodrug approach targets the drug to the liver and bypasses the poor efficiency of the first phosphorylation step that is specifically associated with the uridine nucleoside [4,5]. www.sciencedirect.com
Resistance to nucleotide analogue NS5B inhibitors Go¨tte 105
Mechanisms of action and resistance Each of the approved nucleotide analogue HIV-1 RT inhibitors lack the 30 -hydroxyl group that is essential for the nucleophilic attack on the incoming nucleotide. Hence, once incorporated into the growing DNA chain, these compounds act as chain-terminators. Conversely, the aforementioned nucleotide analogue NS5B inhibitors contain a 30 -hydroxyl group. The mechanism of action is therefore not evident. While the mechanism associated with 40 -azido modified nucleotides remains elusive [8], 20 C-methylated and 20 -a-F-b-C-methylated compounds act as non-obligate chain-terminators [1,9]. Biochemical data have shown that the incorporated inhibitor at the 30 end of the primer prevents binding and or incorporation of the next nucleotide. Modelling studies suggested that the 20 -C-methyl group could conceivably cause a steric conflict with the sugar moiety of the incoming nucleotide substrate. Like HIV-1 RT, HCV NS5B can excise the incorporated inhibitor in the presence of pyrophosphate (PPi) [10]. This reaction can diminish the effect of a given nucleotide analogue in the absence of amino acid substitutions. However, binding of the next nucleotide prevents simultaneous binding of PPi, and provides a certain degree of protection from excision. The incorporated 20 -C-methylcytidine is not protected under these conditions, which supports the notion that the 20 -C-methyl motif can compromise nucleotide binding. Both HIV-1 RT and HCV NS5B can also accept ATP as a pyrophosphate donor [11]. Mutations in HIV-1 RT that decrease susceptibility to certain nucleotide analogues were shown to facilitate the reaction, which provides an important mechanism for resistance [12]. Mutations that affect the excision reaction by HCV NS5B have not been identified; thus, the biological significance of the reverse reaction remains unclear in this context. The S282T mutation in HCV NS5B causes increases in IC50 and EC50 values for 20 -C-methylated compounds. This is also the case for the structurally related 20 -deoxy20 -spirocyclopropylcytidine (TMC647078) [13]. For 20 -Cmethylcytidine, reported increases in IC50 values vary commonly between 5-fold and 20-fold. For 20 -a-F-b-Cmethylcytidine and 20 -a-F-b-C-methyluridine, increases in IC50 values are commonly less pronounced. Similar trends are observed when EC50 values for the corresponding prodrugs mericitabine and sofosbuvir are measured in replicon cells. An interesting mechanistic aspect is that the 20 -a-F-b-C-methylguanosine shows similar potency against wildtype and mutated NS5B, while S282T increases IC50 values of 20 -C-methylguanosine [14,15,16]. The purine base seems to neutralize the effect of S282T in this structural context, which is not the case for pyrimidine-based inhibitors. Prodrugs of 20 -a-Fb-C-methylguanosine were shown to select for a number of mutations at positions 15, 222, 223, 320, and 321. www.sciencedirect.com
Figure 1
Current Opinion in Virology
Location of S282T in HCV NS5B relative to the ultimate base pair. This figure is based on protein data bank (PDB) accession code 4E78, representing the structure of an HCV NS5B enzyme in complex with a short primer/template. The ultimate base pair is shown in white (primer) and green (template). Note that the complex is trapped in the pretranslocated state; thus, the ultimate residue at the primer is here structurally equivalent to an incoming nucleotide. S282T (red) is located at the interface of the two base moieties. The mutation was modelled with the Swiss PDB viewer. G283 (cyan) is located in proximity to the RNA template.
Combinations of these mutations are required to confer significant levels of resistance. Together, these data suggest a more complex interplay between sugar and base moieties in the establishment of a resistant phenotype. The recently reported structures of an engineered genotype 2a HCV NS5B in complex with a short RNA primer/ template substrates provides insight into possible roles plaid by the S282T mutation [16]. The authors suggested that the proximity of S282 to the conserved I160 and G283, that interact with the ultimate base pair, point to a mechanism that may not only involve a steric conflict between S282T and the incoming nucleotide. The side chain of a modelled threonine is located in close proximity to the sugar moiety of an incoming nucleotide, while the adjacent G283 is located in close proximity to the sugar moiety of the complementary template strand (Figure 1). Thus, the mechanism of resistance can also involve altered interaction with the RNA template. S96T, which causes increases in IC50 values for 40 -azidocytidine could affect the positioning of the RNA template. Ser96 is located in the vicinity of the template (Figure 2). Mosley and colleagues speculate that the S96T could move the template towards the nucleotide binding site and reduce the available space for the bulky analogue [16]. Structures of complexes with primers that are terminated with the relevant nucleotide analogues are necessary to validate these models. Current Opinion in Virology 2014, 8:104–108
106 Antivirals and resistance
Figure 2
incorporation of the natural nucleotide when the S282T mutant is compared with the wildtype enzyme [20]. The proximity to the active site helps to explain this observation; however, the detailed underlying mechanism remains elusive.
Current Opinion in Virology
Location of S96 in HCV NS5B relative to the ultimate base pair. S96 (red) is located in close proximity to the template strand, and may affect its precise position at the active site. The colour code is otherwise the same as described for Figure 1.
Clinical significance The barrier to the development of resistance to nucleotide analogue NS5B inhibitors is extremely high when compared to other classes of direct acting antivirals [17]. S96T, that is, the major mutation associated with 40 azidocytidine, has not been identified in clinical phase II trials with the prodrug R1626. Despite the large number of patients enrolled in clinical trials that involved compounds with the 20 -C-methyl-motif or the 20 -a-F-bC-methyl-motif, the S282T mutation was only observed in very few cases. A prominent example is the transient selection of the S282T variant in a patient treated with a combination of sofosbuvir and the HCV NS5A inhibitor ledipasvir [17]. Among other mutations that were identified in HCV NS5A, the S282T mutation in NS5B was detected as a major species (>90%) following 8 weeks of treatment with the two drugs. While the subpopulations of NS5A associated resistant variants remained robust over protracted periods of time, the subpopulation containing S282T declined markedly following removal of the drug. Re-treatment with sofosbuvir, ledipasvir, and ribavirin over 24 weeks resulted in sustained virological response 12 weeks after treatment discontinuation. The S282T mutation has also been identified in a patient who was treated with mericitabine and the ritonavir-boosted protease inhibitor danoprevir [18]. The high barrier to the development of resistance to nucleotide analogue inhibitors has been ascribed to fitness deficits of the mutant variants [2,19,20]. S282T shows only 10–20% of the replication capacity of the wildtype replicon. In reported clinical trials, the S282T mutation was never identified at baseline of untreated individuals, which is in agreement with a significant cost in viral fitness [21]. This interpretation is also consistent with biochemical data that reveal diminished rates of Current Opinion in Virology 2014, 8:104–108
A relatively high genetic barrier poses another problem to the development of resistance to nucleotide analogue. The S282T substitution requires a G to C change at the nucleotide level. Fidelity studies with HCV NS5B have shown that reactions leading to transversion mutations, such as the G to C change, are inefficient when compared with reactions leading to transition mutations [22]. Measurements of frequencies of transitions over transversions revealed 10-fold to 50-fold differences in favour of transition mutations [22,23]. Although the S282T substitution requires only a single nucleotide change, this change seems to be a rare event. Both a limited pool of the S282T variant along with the deficit in replication capacity can compromise the selection of the mutant strain. The low levels of resistance to mericitabine and sofosbuvir may not confer a significant advantage of S282T over the wildtype. Both parameters the relatively high genetic barrier and the significant losses in viral fitness also provide an explanation for the difficulties to select for S96T. Tong and colleagues have recently described the emergence of novel mutations identified in samples partial responders to mericitabine plus peginterferon alfa-2a/ ribavirin treatment [24]. L159F and L320F mutations have been identified in one of 12 partial responders of 405 treated patients. The authors generated mutant replicons Figure 3
Current Opinion in Virology
Location of L159 in HCV NS5B relative to the ultimate base pair. L159 (magenta) is in the centred by S282 (red) and the adjacent residues I160 (yellow) and R158 (blue). I160 and R158 interact with the ultimate base pair at the active site. www.sciencedirect.com
Resistance to nucleotide analogue NS5B inhibitors Go¨tte 107
in attempts to assess the potential effects of these mutations on both replication capacity and resistance to nucleotide analogues. While the single mutations showed diminished replication capacities between 25% and 44% of the wildtype, the double mutant is severely compromised (6% of wildtype). The addition of the S282T mutation generally enhances this effect. The single mutations L159F and L320F, respectively, showed approximately 2-fold increases in EC50 values to mericitabine and sofosbuvir. Combinations of these mutations can cause approximately 4.5-fold increases in EC50 values to mericitabine and approximately 2.5-fold increases in EC50 values to sofosbuvir. This effect is enhanced with the additional presence of S282T. Combinations of L159F/L320F with S282T have not been observed in the clinic, which can be ascribed to the severe losses in viral replication capacity. However, in contrast to S282T and L320F, L159F can be identified in baseline samples. L159 is located next to R158, that can interact with the incoming nucleotide, and I160 that interacts with the template (Figure 3). The structures published by Mosley and colleagues further show that the precise position of these flanking residues also depends on the nature of the ultimate base pair. Hence, the L159F may also exert its effects through altered interaction with the incoming nucleotide analogue and the complementary template.
Conclusion The selection of resistance to nucleoside and nucleotide analogue NS5B inhibitors is associated with high barriers. S282T is only transiently seen in very few clinical cases. The significance of possible alternative pathways that could involve polymorphic sites, for example, L159F, remains to be evaluated. High resolution structures of NS5B in complex with chain-terminated primers are required to provide detailed models of the underlying mechanisms.
Acknowledgements MG is the recipient of a Chercheur National award from the Fonds de la recherche en sante´ du Que´bec (FRSQ). HCV research in his laboratory is currently supported through grants from the Canadian Institutes of Health Research (CIHR), and research contracts from Gilead Sciences Inc., Microbiotix Inc., and Medivir.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
Cortese R et al.: Characterization of resistance to non-obligate chain-terminating ribonucleoside analogs that inhibit hepatitis C virus replication in vitro. J Biol Chem 2003, 278:49164-49170. First report of the S282T mutation in HCV NS5B. 3.
Le Pogam S, Jiang WR, Leveque V, Rajyaguru S, Ma H, Kang H, Jiang S, Singer M, Ali S, Klumpp K, Smith D et al.: In vitro selected Con1 subgenomic replicons resistant to 20 -C-methyl-cytidine or to R1479 show lack of cross resistance. Virology 2006, 351:349-359.
4. Sofia MJ: Nucleotide prodrugs for HCV therapy. Antivir Chem Chemother 2011, 22:23-49. Comprehensive review on nucleoside/nucleotide prodrug approaches. 5. Sofia MJ: Nucleotide prodrugs for the treatment of HCV infection. Adv Pharmacol 2013, 67:39-73. See annotation to Ref. [4]. 6.
Gentile I, Coppola N, Buonomo AR, Zappulo E, Borgia G: Investigational nucleoside and nucleotide polymerase inhibitors and their use in treating hepatitis C virus. Expert Opin Investig Drugs 2014:1-13.
7. Keating GM: Sofosbuvir: a review of its use in patients with chronic hepatitis C. Drugs 2014, 74:1127-1146. Recent review on the clinical use of sofosbuvir. 8.
Klumpp K, Leveque V, Le Pogam S, Ma H, Jiang WR, Kang H, Granycome C, Singer M, Laxton C, Hang JQ, Sarma K et al.: The novel nucleoside analog R1479 (40 -azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture. J Biol Chem 2006, 281:3793-3799.
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Fung A, Jin Z, Dyatkina N, Wang G, Beigelman L, Deval J: Efficiency of incorporation and chain termination determines the inhibition potency of 20 -modified nucleotide analogs against hepatitis C virus polymerase. Antimicrob Agents Chemother 2014, 58:3636-3645.
10. Deval J, Powdrill MH, D’Abramo CM, Cellai L, Gotte M: Pyrophosphorolytic excision of nonobligate chain terminators by hepatitis C virus NS5B polymerase. Antimicrob Agents Chemother 2007, 51:2920-2928. 11. Jin Z, Leveque V, Ma H, Johnson KA, Klumpp K: NTP-mediated nucleotide excision activity of hepatitis C virus RNAdependent rna polymerase. Proc Natl Acad Sci U S A 2013, 110:E348-E357. 12. Menendez-Arias L: Mechanisms of resistance to nucleoside analogue inhibitors of HIV-1 reverse transcriptase. Virus Res 2008, 134:124-146. 13. Berke JM, Vijgen L, Lachau-Durand S, Powdrill MH, Rawe S, Sjuvarsson E, Eriksson S, Gotte M, Fransen E, Dehertogh P, Van den Eynde C et al.: Antiviral activity and mode of action of TMC647078, a novel nucleoside inhibitor of the hepatitis C cirus NS5B polymerase. Antimicrob Agents Chemother 2011, 55:3812-3820. 14. Lam AM, Espiritu C, Bansal S, Micolochick Steuer HM, Zennou V, Otto MJ, Furman PA: Hepatitis C virus nucleotide inhibitors PSI352938 and PSI-353661 exhibit a novel mechanism of resistance requiring multiple mutations within replicon RNA. J Virol 2011, 85:12334-12342. 15. Lam AM, Espiritu C, Murakami E, Zennou V, Bansal S, Micolochick Steuer HM, Niu C, Keilman M, Bao H, Bourne N, Veselenak RL et al.: Inhibition of hepatitis C virus replicon RNA synthesis by PSI-352938, a cyclic phosphate prodrug of beta-d-20 -deoxy-20 alpha-fluoro-20 -beta-c-methylguanosine. Antimicrob Agents Chemother 2011, 55:2566-2575.
Carroll SS, Tomassini JE, Bosserman M, Getty K, Stahlhut MW, Eldrup AB, Bhat B, Hall D, Simcoe AL, LaFemina R, Rutkowski CA et al.: Inhibition of hepatitis C virus RNA replication by 20 modified nucleoside analogs. J Biol Chem 2003, 278: 11979-11984. First study on 20 -C-methylated nucleotide analogue inhibitors of HCV NS5B.
16. Mosley RT, Edwards TE, Murakami E, Lam AM, Grice RL, Du J, Sofia MJ, Furman PA, Otto MJ: Structure of hepatitis C virus polymerase in complex with primer-template RNA. J Virol 2012, 86:6503-6511. Structure of a binary complex of HCV NS5B with primer/template. This structure is highly relevant to a better understanding of mechanisms of action and reistance to nucleotide analogue inhibitors.
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17. a. Vermehren J, Sarrazin C: The role of resistance in HCV treatment. Best Pract Res Clin Gastroenterol 2012, 26:487-503;
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Migliaccio G, Tomassini JE, Carroll SS, Tomei L, Altamura S, Bhat B, Bartholomew L, Bosserman MR, Ceccacci A, Colwell LF,
www.sciencedirect.com
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b. Mo H: HCV nucleoside NS5B polymerase inhibitors: antiviral activities and barriers to resistance among different HCV genotype and subtypes. Antivir Therapy 2014, 19(Suppl. 1):A21. 18. Tong X, Li L, Haines K, Najera I: Identification of the NS5B S282T resistant variant and two novel amino acid substitutions that affect replication capacity in hepatitis C virus-infected patients treated with mericitabine and danoprevir. Antimicrob Agents Chemother 2014, 58:3105-3114. Report of the emergence of S282T in vivo. 19. Ali S, Leveque V, Le Pogam S, Ma H, Philipp F, Inocencio N, Smith M, Alker A, Kang H, Najera I, Klumpp K et al.: Selected replicon variants with low-level in vitro resistance to the hepatitis C virus NS5B polymerase inhibitor PSI-6130 lack cross-resistance with R1479. Antimicrob Agents Chemother 2008, 52:4356-4369. 20. Dutartre H, Bussetta C, Boretto J, Canard B: General catalytic deficiency of hepatitis C virus RNA polymerase with an S282T mutation and mutually exclusive resistance towards 20 modified nucleotide analogues. Antimicrob Agents Chemother 2006, 50:4161-4169.
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21. Pawlotsky JM, Najera I, Jacobson I: Resistance to mericitabine, a nucleoside analogue inhibitor of HCV RNA-dependent RNA polymerase. Antivir Therapy 2012, 17:411-423. 22. Powdrill MH, Tchesnokov EP, Kozak RA, Russell RS, Martin R, Svarovskaia ES, Mo H, Kouyos RD, Gotte M: Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance. Proc Natl Acad Sci U S A 2011, 108:20509-20513. Characterization of HCV NS5B fidelity. 23. Ribeiro RM, Li H, Wang S, Stoddard MB, Learn GH, Korber BT, Bhattacharya T, Guedj J, Parrish EH, Hahn BH, Shaw GM et al.: Quantifying the diversification of hepatitis C virus (HCV) during primary infection: estimates of the in vivo mutation rate. PLoS Pathog 2012, 8:e1002881. 24. Tong X, Le Pogam S, Li L, Haines K, Piso K, Baronas V, Yan JM, So SS, Klumpp K, Najera I: In vivo emergence of a novel mutant L159F/L320F in the NS5B polymerase confers low-level resistance to the HCV polymerase inhibitors mericitabine and sofosbuvir. J Infect Dis 2014, 209:668-675. Identification of possible alternative resistance pathways to nucleotide analogue HCV NS5B inhibitors.
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ScienceDirect Resistance to nucleotide analogue inhibitors of hepatitis C virus NS5B: mechanisms and clinical relevance Matthias Go¨tte1,2,3 The high barrier to the development of resistance to nucleotide analogue inhibitors of the hepatitis C virus (HCV) RNAdependent RNA polymerase is an intriguing property of this class of drugs. The S282T substitution in the viral polymerase confers resistance to 20 -C-methylated nucleotide analogues. Although this mutation can be selected in HCV replicons, it has only been identified in very few cases in the clinic. Alternative resistance pathways are likewise rarely seen in vivo. Possible underlying mechanisms that are associated with the selection and establishment of a resistant genotype are discussed in this review. Addresses 1 Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec H3A 2B4, Canada 2 Department of Biochemistry, McGill University, 3655 Sir William Osler Promenade, Montreal, Quebec H3G 1Y6, Canada 3 Department of Medicine, Division of Experimental Medicine, McGill University, 1110 Pine Avenue West, Montreal, Quebec H3A 1A3, Canada Corresponding author: Go¨tte, Matthias ([email protected])
Current Opinion in Virology 2014, 8:104–108 This review comes from a themed issue on Antivirals and resistance
(HCV) RNA-dependent RNA polymerase or non-structural protein 5B (NS5B).
Development of nucleotide analogue NS5B inhibitors In 2003, Carroll and colleagues reported that the triphosphate form of 20 -C-methylated and 20 -O-methylated nucleoside analogues are efficient inhibitors of HCV NS5B [1]. These compounds also showed antiviral activity in subgenomic replicon cells. 20 -C-methyladenosine was more potent than 20 -O-methylcytidine, which has been ascribed to higher intracellular concentrations of 20 C-methyladenosine triphosphate. Subsequently, Migliaccio and colleagues reported the selection of resistant replicons with a single S282T mutation HCV NS5B [2]. The isolated mutant variant also conferred decreased susceptibility to 20 -C-methylguanosine. Another chemical class of nucleotide analogues that contain the 40 -azido modification select for the S96T substitution in NS5B [3]. Cross-resistance between the two different classes of NS5B inhibitors is commonly not observed. Both 20 -methyl-modified and 40 -azido-modified compounds show generally pan-genotypic activity.
Edited by Luis Mene´ndez-Arias and Douglas D Richman
http://dx.doi.org/10.1016/j.coviro.2014.07.010 1879-6257/Published by Elsevier B.V.
Various prodrug approaches were developed to increase bioavailability and to address limitations associated with the intracellular conversion to the active triphosphate form [4,5]. NM283, a prodrug of 20 -C-methylcytidine, was the first nucleotide analogue under clinical investigation for treatment of HCV infection. However, toxicities and/or limited potency caused the discontinuation of several of these first-generation nucleotide analogues, including NM283 and R1626, that is, the prodrug of 40 -Cazidocytidine.
Introduction Nucleotide analogue inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) remain important components in currently available treatment regimens. The diverse and often complex resistance patterns associated with the use of these drugs have been extensively described. Alternative genetic pathways that can involve either single or multiple amino acid substitutions in HIV-1 RT also provide various defined biochemical mechanisms. These previous findings raised the question whether similar diverse and complex resistance patterns may also evolve in the context of novel nucleotide-based therapies against chronic hepatitis C infection. The purpose of this review is to discuss nature, mechanistic aspects, and relevance of resistance to nucleotide analogue inhibitors of the hepatitis C virus Current Opinion in Virology 2014, 8:104–108
Next generations of nucleotide analogues include mericitabine and sofosbuvir. Mericitabine is currently evaluated in advanced clinical trials [6], while sofosbuvir is already approved for the treatment of chronic hepatitis C infection [7]. Both drugs share the 20 -a-F-b-C-methyl motif of the sugar, and differ in the nature of their base moiety and prodrug approach. Mericitabine is a 20 -a-F-bC-methylcytidine with a hydrolysable 30 , 50 -diester moiety. Sofosbuvir is a 20 -a-F-b-C-methyluridine with a phosphoramidate that, following hydrolysis, yields the 50 -monophosphate of 20 -a-F-b-C-methyluridine. This prodrug approach targets the drug to the liver and bypasses the poor efficiency of the first phosphorylation step that is specifically associated with the uridine nucleoside [4,5]. www.sciencedirect.com
Resistance to nucleotide analogue NS5B inhibitors Go¨tte 105
Mechanisms of action and resistance Each of the approved nucleotide analogue HIV-1 RT inhibitors lack the 30 -hydroxyl group that is essential for the nucleophilic attack on the incoming nucleotide. Hence, once incorporated into the growing DNA chain, these compounds act as chain-terminators. Conversely, the aforementioned nucleotide analogue NS5B inhibitors contain a 30 -hydroxyl group. The mechanism of action is therefore not evident. While the mechanism associated with 40 -azido modified nucleotides remains elusive [8], 20 C-methylated and 20 -a-F-b-C-methylated compounds act as non-obligate chain-terminators [1,9]. Biochemical data have shown that the incorporated inhibitor at the 30 end of the primer prevents binding and or incorporation of the next nucleotide. Modelling studies suggested that the 20 -C-methyl group could conceivably cause a steric conflict with the sugar moiety of the incoming nucleotide substrate. Like HIV-1 RT, HCV NS5B can excise the incorporated inhibitor in the presence of pyrophosphate (PPi) [10]. This reaction can diminish the effect of a given nucleotide analogue in the absence of amino acid substitutions. However, binding of the next nucleotide prevents simultaneous binding of PPi, and provides a certain degree of protection from excision. The incorporated 20 -C-methylcytidine is not protected under these conditions, which supports the notion that the 20 -C-methyl motif can compromise nucleotide binding. Both HIV-1 RT and HCV NS5B can also accept ATP as a pyrophosphate donor [11]. Mutations in HIV-1 RT that decrease susceptibility to certain nucleotide analogues were shown to facilitate the reaction, which provides an important mechanism for resistance [12]. Mutations that affect the excision reaction by HCV NS5B have not been identified; thus, the biological significance of the reverse reaction remains unclear in this context. The S282T mutation in HCV NS5B causes increases in IC50 and EC50 values for 20 -C-methylated compounds. This is also the case for the structurally related 20 -deoxy20 -spirocyclopropylcytidine (TMC647078) [13]. For 20 -Cmethylcytidine, reported increases in IC50 values vary commonly between 5-fold and 20-fold. For 20 -a-F-b-Cmethylcytidine and 20 -a-F-b-C-methyluridine, increases in IC50 values are commonly less pronounced. Similar trends are observed when EC50 values for the corresponding prodrugs mericitabine and sofosbuvir are measured in replicon cells. An interesting mechanistic aspect is that the 20 -a-F-b-C-methylguanosine shows similar potency against wildtype and mutated NS5B, while S282T increases IC50 values of 20 -C-methylguanosine [14,15,16]. The purine base seems to neutralize the effect of S282T in this structural context, which is not the case for pyrimidine-based inhibitors. Prodrugs of 20 -a-Fb-C-methylguanosine were shown to select for a number of mutations at positions 15, 222, 223, 320, and 321. www.sciencedirect.com
Figure 1
Current Opinion in Virology
Location of S282T in HCV NS5B relative to the ultimate base pair. This figure is based on protein data bank (PDB) accession code 4E78, representing the structure of an HCV NS5B enzyme in complex with a short primer/template. The ultimate base pair is shown in white (primer) and green (template). Note that the complex is trapped in the pretranslocated state; thus, the ultimate residue at the primer is here structurally equivalent to an incoming nucleotide. S282T (red) is located at the interface of the two base moieties. The mutation was modelled with the Swiss PDB viewer. G283 (cyan) is located in proximity to the RNA template.
Combinations of these mutations are required to confer significant levels of resistance. Together, these data suggest a more complex interplay between sugar and base moieties in the establishment of a resistant phenotype. The recently reported structures of an engineered genotype 2a HCV NS5B in complex with a short RNA primer/ template substrates provides insight into possible roles plaid by the S282T mutation [16]. The authors suggested that the proximity of S282 to the conserved I160 and G283, that interact with the ultimate base pair, point to a mechanism that may not only involve a steric conflict between S282T and the incoming nucleotide. The side chain of a modelled threonine is located in close proximity to the sugar moiety of an incoming nucleotide, while the adjacent G283 is located in close proximity to the sugar moiety of the complementary template strand (Figure 1). Thus, the mechanism of resistance can also involve altered interaction with the RNA template. S96T, which causes increases in IC50 values for 40 -azidocytidine could affect the positioning of the RNA template. Ser96 is located in the vicinity of the template (Figure 2). Mosley and colleagues speculate that the S96T could move the template towards the nucleotide binding site and reduce the available space for the bulky analogue [16]. Structures of complexes with primers that are terminated with the relevant nucleotide analogues are necessary to validate these models. Current Opinion in Virology 2014, 8:104–108
106 Antivirals and resistance
Figure 2
incorporation of the natural nucleotide when the S282T mutant is compared with the wildtype enzyme [20]. The proximity to the active site helps to explain this observation; however, the detailed underlying mechanism remains elusive.
Current Opinion in Virology
Location of S96 in HCV NS5B relative to the ultimate base pair. S96 (red) is located in close proximity to the template strand, and may affect its precise position at the active site. The colour code is otherwise the same as described for Figure 1.
Clinical significance The barrier to the development of resistance to nucleotide analogue NS5B inhibitors is extremely high when compared to other classes of direct acting antivirals [17]. S96T, that is, the major mutation associated with 40 azidocytidine, has not been identified in clinical phase II trials with the prodrug R1626. Despite the large number of patients enrolled in clinical trials that involved compounds with the 20 -C-methyl-motif or the 20 -a-F-bC-methyl-motif, the S282T mutation was only observed in very few cases. A prominent example is the transient selection of the S282T variant in a patient treated with a combination of sofosbuvir and the HCV NS5A inhibitor ledipasvir [17]. Among other mutations that were identified in HCV NS5A, the S282T mutation in NS5B was detected as a major species (>90%) following 8 weeks of treatment with the two drugs. While the subpopulations of NS5A associated resistant variants remained robust over protracted periods of time, the subpopulation containing S282T declined markedly following removal of the drug. Re-treatment with sofosbuvir, ledipasvir, and ribavirin over 24 weeks resulted in sustained virological response 12 weeks after treatment discontinuation. The S282T mutation has also been identified in a patient who was treated with mericitabine and the ritonavir-boosted protease inhibitor danoprevir [18]. The high barrier to the development of resistance to nucleotide analogue inhibitors has been ascribed to fitness deficits of the mutant variants [2,19,20]. S282T shows only 10–20% of the replication capacity of the wildtype replicon. In reported clinical trials, the S282T mutation was never identified at baseline of untreated individuals, which is in agreement with a significant cost in viral fitness [21]. This interpretation is also consistent with biochemical data that reveal diminished rates of Current Opinion in Virology 2014, 8:104–108
A relatively high genetic barrier poses another problem to the development of resistance to nucleotide analogue. The S282T substitution requires a G to C change at the nucleotide level. Fidelity studies with HCV NS5B have shown that reactions leading to transversion mutations, such as the G to C change, are inefficient when compared with reactions leading to transition mutations [22]. Measurements of frequencies of transitions over transversions revealed 10-fold to 50-fold differences in favour of transition mutations [22,23]. Although the S282T substitution requires only a single nucleotide change, this change seems to be a rare event. Both a limited pool of the S282T variant along with the deficit in replication capacity can compromise the selection of the mutant strain. The low levels of resistance to mericitabine and sofosbuvir may not confer a significant advantage of S282T over the wildtype. Both parameters the relatively high genetic barrier and the significant losses in viral fitness also provide an explanation for the difficulties to select for S96T. Tong and colleagues have recently described the emergence of novel mutations identified in samples partial responders to mericitabine plus peginterferon alfa-2a/ ribavirin treatment [24]. L159F and L320F mutations have been identified in one of 12 partial responders of 405 treated patients. The authors generated mutant replicons Figure 3
Current Opinion in Virology
Location of L159 in HCV NS5B relative to the ultimate base pair. L159 (magenta) is in the centred by S282 (red) and the adjacent residues I160 (yellow) and R158 (blue). I160 and R158 interact with the ultimate base pair at the active site. www.sciencedirect.com
Resistance to nucleotide analogue NS5B inhibitors Go¨tte 107
in attempts to assess the potential effects of these mutations on both replication capacity and resistance to nucleotide analogues. While the single mutations showed diminished replication capacities between 25% and 44% of the wildtype, the double mutant is severely compromised (6% of wildtype). The addition of the S282T mutation generally enhances this effect. The single mutations L159F and L320F, respectively, showed approximately 2-fold increases in EC50 values to mericitabine and sofosbuvir. Combinations of these mutations can cause approximately 4.5-fold increases in EC50 values to mericitabine and approximately 2.5-fold increases in EC50 values to sofosbuvir. This effect is enhanced with the additional presence of S282T. Combinations of L159F/L320F with S282T have not been observed in the clinic, which can be ascribed to the severe losses in viral replication capacity. However, in contrast to S282T and L320F, L159F can be identified in baseline samples. L159 is located next to R158, that can interact with the incoming nucleotide, and I160 that interacts with the template (Figure 3). The structures published by Mosley and colleagues further show that the precise position of these flanking residues also depends on the nature of the ultimate base pair. Hence, the L159F may also exert its effects through altered interaction with the incoming nucleotide analogue and the complementary template.
Conclusion The selection of resistance to nucleoside and nucleotide analogue NS5B inhibitors is associated with high barriers. S282T is only transiently seen in very few clinical cases. The significance of possible alternative pathways that could involve polymorphic sites, for example, L159F, remains to be evaluated. High resolution structures of NS5B in complex with chain-terminated primers are required to provide detailed models of the underlying mechanisms.
Acknowledgements MG is the recipient of a Chercheur National award from the Fonds de la recherche en sante´ du Que´bec (FRSQ). HCV research in his laboratory is currently supported through grants from the Canadian Institutes of Health Research (CIHR), and research contracts from Gilead Sciences Inc., Microbiotix Inc., and Medivir.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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