Selection of HIV-1 for resistance to fifth-generation protease inhibitors reveals two independent pathways to high-level resistance
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This manuscript significantly advances our understanding of the development of drug resistance in the HIV-1 protease. The paper addresses the fundamental relationship between resistance mutations and inhibitor structure and will be useful in the design and development of the next generation of inhibitors.
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Abstract
Darunavir (DRV) is exceptional among potent HIV-1 protease inhibitors (PIs) in high drug concentrations that are achieved in vivo. Little is known about the de novo resistance pathway for DRV. We selected for resistance to high drug concentrations against 10 PIs and their structural precursor DRV. Mutations accumulated through two pathways (anchored by protease mutations I50V or I84V). Small changes in the inhibitor P1'-equivalent position led to preferential use of one pathway over the other. Changes in the inhibitor P2'-equivalent position determined differences in potency that were retained in the resistant viruses and that impacted the selected mutations. Viral variants from the two pathways showed differential selection of compensatory mutations in Gag cleavage sites. These results reveal the high level of selective pressure that is attainable with fifth-generation PIs and how features of the inhibitor affect both the resistance pathway and the residual potency in the face of resistance.
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eLife assessment
This manuscript significantly advances our understanding of the development of drug resistance in the HIV-1 protease. The paper addresses the fundamental relationship between resistance mutations and inhibitor structure and will be useful in the design and development of the next generation of inhibitors.
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Reviewer #1 (Public Review):
The authors used viral replication assays to select for and define the resistance pathways against ten developmental Protease Inhibitors (PIs) and their parent drug, Darunavir (DRV), which is one of the leading antiretrovirals used to treat people living with HIV/AIDS. There are two specific regions of the small molecule inhibitors that are actively being modified to increase potency against drug-resistant mutants, the P1' region, and the P2' region, which protrude into pockets of PR occupied by I84 / I50, and a neighboring region containing D29-D30, respectively. Selections using drugs containing small modifications of the P1' region led to primary mutations at PR position I184V, but not I150V. In contrast, selections using drugs containing larger modifications at the P1' region led to primary mutations at …
Reviewer #1 (Public Review):
The authors used viral replication assays to select for and define the resistance pathways against ten developmental Protease Inhibitors (PIs) and their parent drug, Darunavir (DRV), which is one of the leading antiretrovirals used to treat people living with HIV/AIDS. There are two specific regions of the small molecule inhibitors that are actively being modified to increase potency against drug-resistant mutants, the P1' region, and the P2' region, which protrude into pockets of PR occupied by I84 / I50, and a neighboring region containing D29-D30, respectively. Selections using drugs containing small modifications of the P1' region led to primary mutations at PR position I184V, but not I150V. In contrast, selections using drugs containing larger modifications at the P1' region led to primary mutations at PR position I150V, a pathway that is less fit. Furthermore, having modifications at the P2' position added additional potency to the inhibitors, most evident within the I184V pathway. The authors rationalize their findings using previously published structural biology data. These results provide the first evidence for de novo pathway selection using state-of-the-art drugs based on the DRV scaffold and provide an atomic basis for designing compounds that are highly active against DRMs. The comprehensive nature of the analysis of drug resistance to the latest generation PIs, and the insights gained that can be rationalized based on atomic structure, are the major strengths of the paper. The weakness is the lack of commentary on the accessory mutations, which frequently arise in the selections but are not well-explained. It would also be useful to provide some concrete suggestions for minimizing drug resistance using 5th generation PIs, as part of a discussion.
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Reviewer #2 (Public Review):
Spielvogel and colleagues report in vitro studies investigating the development of de novo resistance of HIV to Darunavir. Darunavir is one of the most widely used protease inhibitors worldwide, but pathways for the development of de novo resistance are uncertain, as many individuals have had prior protease inhibitor experience prior to treatment with darunavir. As such studies of the kind reported here are essential. The authors have performed foundational studies using compelling and complementary approaches to characterize the emergence of protease drug resistance. They have investigated darunavir, as well as a series of 10 structurally related compounds to provide a clear picture of the role of side chains in the development of resistance. They have complemented these studies with precise structural …
Reviewer #2 (Public Review):
Spielvogel and colleagues report in vitro studies investigating the development of de novo resistance of HIV to Darunavir. Darunavir is one of the most widely used protease inhibitors worldwide, but pathways for the development of de novo resistance are uncertain, as many individuals have had prior protease inhibitor experience prior to treatment with darunavir. As such studies of the kind reported here are essential. The authors have performed foundational studies using compelling and complementary approaches to characterize the emergence of protease drug resistance. They have investigated darunavir, as well as a series of 10 structurally related compounds to provide a clear picture of the role of side chains in the development of resistance. They have complemented these studies with precise structural studies of the interactions of drug with WT and mutant viruses. These data are relevant to the understanding of clinical responses to darunavir and are important in developing new protease inhibitors.
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Reviewer #3 (Public Review):
Darunavir (DRV) has been shown to be a potent HIV-1 protease inhibitor in individuals, has pM binding to the protease active site, has activity to protease inhibitor resistant HIV-1s, and has been reported to be difficult to develop resistance to individuals and in tissue culture. The authors argue that given published studies of generating HIV-1 resistance to DRV in tissue culture was not accomplished and all published studies started with either a drug-resistant virus or a combination of drug-resistant viruses for selection, new information can be gleaned as to the viral mutational pathways leading to drug-resistant viruses from HIV-1 wild type (no pre-existing drug mutations) NL4-3.
To better understand the development of HIV-1 wild-type DRV resistance, Spielvogel and colleagues detail their studies on …
Reviewer #3 (Public Review):
Darunavir (DRV) has been shown to be a potent HIV-1 protease inhibitor in individuals, has pM binding to the protease active site, has activity to protease inhibitor resistant HIV-1s, and has been reported to be difficult to develop resistance to individuals and in tissue culture. The authors argue that given published studies of generating HIV-1 resistance to DRV in tissue culture was not accomplished and all published studies started with either a drug-resistant virus or a combination of drug-resistant viruses for selection, new information can be gleaned as to the viral mutational pathways leading to drug-resistant viruses from HIV-1 wild type (no pre-existing drug mutations) NL4-3.
To better understand the development of HIV-1 wild-type DRV resistance, Spielvogel and colleagues detail their studies on characterizing HIV-1 protease genomic and structural alterations and viral fitness before and during the development of tissue culture resistance to DRV, as well as 10 new compounds (UMass compound series) based on DRV. The UMass compounds have distinct R1 and R2 groups as compared to DRV, which provides for a comprehensive chemical toolset to probe protease genetics and structural changes and alterations in viral fitness resulting during HIV protease drug resistance development in tissue culture. Differences in HIV protease resistance patterns developing over time combined with the potency of the protease inhibitors to HIV mutants resulting from inhibitor selections provide insights as to how DRV chemical groups impact resistance development. The manuscript is comprehensive, well-written, and informative, yet dense and with some figures that readers may not find informative.
Protease inhibitor tissue culture selection of wild-type NL4-3 was based on increasing protease inhibitor concentrations over time. Generally, the DRV resistance mutations that came up early de novo from wild-type NL4-3 virus were, 84V, followed by the acquisition of accessory mutations, predominately 54L and 82I, with 84V, 85V, 46I, 47V, 63P, and others as well, which became entrenched over time. The 84V mutational series have been reported for DRV as the authors noted. To determine the DRV selection pattern from pre-existing HIV single drug-resistant population a pool of 26 single mutant viruses was used for selection. Similar patterns were seen as for wild-type viruses, starting with 84V.
Interestingly, when the UMass compound series was used to select wild-type NL4-3 in tissue culture, 3 mutational series resulted, a protease mutational pattern similar to DRV (UMass 1, and 4, a protease mutational pattern starting with 50V, and followed by the predominate accessory mutations 10F, 13V, 33F, 46I, 63P, and 71V, but not 84V (UMass 3,6,7,8,9, and 10) and a mixture of both populations (UMass 2 and 5). When the HIV single drug-resistant population pool was used, which didn't contain 50V, was used for selection, UMass 2,4,7, and 8 retained the same mutational patterns as the original wild-type HIV selection, where, interestingly, UMass 6 utilized the 84V mutational pathway, rather than 50V, when the 84V mutation was pre-existing.
The results pointed out that modification of the DRV R2 and R1 groups alters selection patterns. It appears that a smaller hydrophobic side chain at the P1' position appears to drive towards 84V selection, whereas a larger side chain selects for the 50V pathway. UMass compounds 2, 5, 7, and 10 demonstrate the highest potency to both 50V/71V and 84V mutant viruses. Interestingly, UMass 2 and 5 were selected for both 50V/71V and 84V resistance mutational pathways, whereas 7 and 10 were selected for 50V/71V pathways.
Based on entry/replication studies, the authors argue that pushing viruses to select 50V/71V mutational pathways in protease, vs 84V mutational pathways in protease, promotes a higher genetic barrier to overcome resistance. This would be due to the reduction in fitness for the 50V/71V protease mutant and the large number of accessory mutants required to regain fitness. However, more in-depth analyses of the various mutants are warranted to support this point, such as head-to-head viral replication studies. A further limitation to the general conclusions is whether mutations in Gag provide for compensatory mutations to augment protease (and viral) fitness for the UMass compound findings.
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