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    • Under increased drug pressure more protease

    2021-12-01

    Under increased drug pressure, more protease variants with more than one substitution will likely become clinically relevant. The accumulation of additional substitutions can allow RAS variants to emerge that alone are not viable, but in combination can rescue the viral fitness. We previously demonstrated that inhibitors with a P2–P4 macrocycle are highly susceptible to substitutions at Ala156, as a change to a larger side chain results in steric clashes with the inhibitor's macrocycle. Ala156 substitutions cause low replicative capacity, but additional changes at other positions in the NS3/4A protease can improve enzymatic activity and thus viral fitness, leading to clinically relevant variants. Voxilaprevir and glecaprevir also select for substitutions at Ala156 in vitro, which causes a large fold shift in inhibitor potency. Moreover, Ala156-Asp168 double substitutions have been selected in vitro, which improve fitness. Although not yet observed clinically, the A156T substitution if coupled with such a fitness-rescuing second substitution could cause resistance to all P2–P4 macrocyclic PIs with a P2 quinoxaline moiety. In fact, the additional substitution does not have to occur at the active site. We have shown in HIV-1 protease that active-site and distal substitutions often occur in combination to confer resistance (Ragland et al., 2014). Similarly, glecaprevir selected substitutions at Ala156 in combination with Gln/Pro89 in GT1a/b, which is located outside of the active site. This additional substitution at position 89 appears to have improved replicative efficiency to 100% (Ng et al., 2017). Understanding the molecular mechanisms of resistance and enzymatic SID 26681509 of these multi-substituted variants will be necessary to improve the potency of PIs against emerging resistant variants.
    STAR★Methods
    Acknowledgments This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. We thank beamline specialists at 23-ID-B for their help in data collection. This work was supported by a grant from the National Institute of Allergy and Infectious Diseases of the NIH (R01 AI085051). A.N.M. was also supported by the National Institute of General Medical Sciences of the NIH (F31 GM119345). We thank Profs. Brian Kelch, Paul Thompson, William Royer, and Dan Bolon for helpful discussions.
    Introduction Regimens based on direct-acting antivirals (DAAs) have revolutionized treatment of patients with chronic infection with hepatitis C virus (HCV), which globally has been estimated to cause 70–150 million chronic infections and at least 400,000 deaths annually.[1], [2] Approved DAAs target the HCV protease (NS3P), NS5A and NS5B.[3], [4] Protease inhibitors (PIs), available since 2011, constitute an important component of DAA-based combination therapies.[3], [4], [5] While the initially developed PIs telaprevir and boceprevir have been discontinued, simeprevir might be used for treatment of patients infected with genotype 1, and grazoprevir or paritaprevir for genotypes 1 and 4.[6], [7] Asunaprevir, approved in Asia and the Middle East, is used only for subtype 1b. The novel PIs glecaprevir and voxilaprevir are recommended for genotypes 1-6.[6], [7] A subset of DAA-treated patients experience treatment failure, which is associated with selection of resistance-associated substitutions (RASs) that are either naturally occurring and pre-existing at baseline or rapidly acquired during treatment.[3], [4] Several clinical studies have provided evidence that baseline RASs in DAA targets can compromise DAA treatment efficacy.[4], [5] The prevalence of pre-existing RASs depends on their effect on viral fitness, influencing persistence and spread in human populations. Of described naturally occurring NS3P RASs, Q80K shows the highest prevalence: for genotype 1a-infected patients, 19%, 48% and 9% are carrying Q80K in Europe, North America, and South America, respectively.[3], [8] Strong regional deviations in Q80K prevalence have been observed, with European prevalence rates ranging from 5% in Norway to 75% in Poland. Q80K is rarely observed for genotype 1b and for other genotypes limited data are available. For genotypes 1a and 1b, Q80K and the less prevalent Q80R caused resistance to simeprevir in vitro,[9], [10], [11] and for 1a, baseline Q80K resulted in decreased efficacy of simeprevir-based treatment regimens.[4], [5] For other genotypes, data on the effect of Q80K on PI efficacy are not available. While PI RASs can confer extensive cross-resistance among different PIs, little is known about the impact of Q80K on the efficacy of PIs other than simeprevir. Finally, molecular mechanisms underlying treatment failure mediated by baseline RASs have not been studied.