Selection of HIV-1 for Resistance to Fifth Generation Protease Inhibitors Reveals Two Independent Pathways to High-Level Resistance

Abstract

23Well-designed viral protease inhibitors (PIs) potently inhibit replication as well as create 24 a high genetic barrier for resistance. Through in vivo selective pressure, we have generated 25 high-level resistance against ten HIV-1 PIs and their precursor, the FDA-approved drug 26 darunavir (DRV), achieving 1,000-fold resistance over the starting EC50. The accumulation of 27 mutations revealed two pathways to high-level resistance, resulting in protease variants with up 28 to 14 mutations in and outside of the active site. The two pathways demonstrate the interplay 29 between drug resistance and viral fitness. Replicate selections showed that one inhibitor could 30 select for resistance through either pathway, although subtle changes in chemical structure of 31 the inhibitors led to preferential use of one pathway over the other. Viral variants from the two 32 pathways showed differential selection of compensatory mutations in Gag cleavage sites. These 33 results reveal the high-level of selective pressure that is attainable with these fourth-generation 34 protease inhibitors, and the interplay between selection of mutations to confer resistance while 35 maintaining viral fitness. 36 37 3 Introduction 38

Deciphering Complex Mechanisms of Resistance and Loss of Potency through Coupled Molecular Dynamics and Machine Learning

“…26,27 Most substitutions in these resistant variants are distal from the inhibitor binding site. S1) Cluster 3 included all variants obtained from viral passaging experiments with DRV and DRV analogs 14 . Cluster 2 included variants with less than 5 amino acid substitutions.…”

“…HIV-1 protease variants and experimental inhibitor potency Drug resistant variants of HIV-1 protease were obtained from viral passaging experiments with DRV and closely related analogs. 50 These variants were supplemented with variants bearing known primary active site mutations. The crystal structures of wild type protease, variants with primary resistance mutations, and highly drug resistant variants bound to DRV had been determined previously.…”

“…In one crystal structure (3ucb; Table 1) a second molecule of DRV was co-crystallized outside of the active site and was removed for this analysis. All variants from viral passaging (Source: 50 , Table1) that lacked experimental structures were modeled using Prime. 53,54 Structures were prepared using the Schrödinger Protein Preparation Wizard.…”

“…Resistance to DRV still emerges through accumulation of multiple mutations both proximal and distal from the active site. [11][12][13][14] Elucidating the resistance mechanism for very potent inhibitors, such as the HIV-1 protease inhibitor DRV, provides insights into how amino acid substitutions alter the structure, dynamics and function of a therapeutic target. 15,16 However the molecular mechanisms by which mutations, in particular those distal from the active site, confer resistance remains elusive.…”

Deciphering complex mechanisms of resistance and loss of potency through coupled molecular dynamics and machine learning.

“…The protease variants clustered into 3 major subgroups based on amino acid sequence. (Fig 1C, Table S1) Cluster 3 included all variants obtained from viral passaging experiments with DRV and DRV analogs 14 . Cluster 2 included variants with less than 5 amino acid substitutions.…”