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Structural basis of second-generation HIV integrase inhibitor action and viral resistance.

ABSTRACT: Although second-generation HIV integrase strand-transfer inhibitors (INSTIs) are prescribed throughout the world, the mechanistic basis for the superiority of these drugs is poorly understood. We used single-particle cryo-electron microscopy to visualize the mode of action of the advanced INSTIs dolutegravir and bictegravir at near-atomic resolution. Glutamine-148→histidine (Q148H) and glycine-140→serine (G140S) amino acid substitutions in integrase that result in clinical INSTI failure perturb optimal magnesium ion coordination in the enzyme active site. The expanded chemical scaffolds of second-generation compounds mediate interactions with the protein backbone that are critical for antagonizing viruses containing the Q148H and G140S mutations. Our results reveal that binding to magnesium ions underpins a fundamental weakness of the INSTI pharmacophore that is exploited by the virus to engender resistance and provide a structural framework for the development of this class of anti-HIV/AIDS therapeutics.

SUBMITTER: Cook NJ

PROVIDER: S-EPMC7023979 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Structural basis of second-generation HIV integrase inhibitor action and viral resistance.

Cook Nicola J NJ Li Wen W Berta Dénes D Badaoui Magd M Ballandras-Colas Allison A Nans Andrea A Kotecha Abhay A Rosta Edina E Engelman Alan N AN Cherepanov Peter P

Science (New York, N.Y.) 20200130 6479

Although second-generation HIV integrase strand-transfer inhibitors (INSTIs) are prescribed throughout the world, the mechanistic basis for the superiority of these drugs is poorly understood. We used single-particle cryo-electron microscopy to visualize the mode of action of the advanced INSTIs dolutegravir and bictegravir at near-atomic resolution. Glutamine-148→histidine (Q148H) and glycine-140→serine (G140S) amino acid substitutions in integrase that result in clinical INSTI failure perturb ...[more]

PMID: 32001525

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Project description:A betulinic acid-based compound, bevirimat (BVM), inhibits HIV-1 maturation by blocking a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. Previous studies showed that mutations conferring resistance to BVM cluster around the CA-SP1 cleavage site. Single amino acid polymorphisms in the SP1 region of Gag and the C terminus of CA reduced HIV-1 susceptibility to BVM, leading to the discontinuation of BVM's clinical development. We recently reported a series of "second-generation" BVM analogs that display markedly improved potency and breadth of activity relative to the parent molecule. Here, we demonstrate that viral clones bearing BVM resistance mutations near the C terminus of CA are potently inhibited by second-generation BVM analogs. We performed de novo selection experiments to identify mutations that confer resistance to these novel compounds. Selection experiments with subtype B HIV-1 identified an Ala-to-Val mutation at SP1 residue 1 and a Pro-to-Ala mutation at CA residue 157 within the major homology region (MHR). In selection experiments with subtype C HIV-1, we identified mutations at CA residue 230 (CA-V230M) and SP1 residue 1 (SP1-A1V), residue 5 (SP1-S5N), and residue 10 (SP1-G10R). The positions at which resistance mutations arose are highly conserved across multiple subtypes of HIV-1. We demonstrate that the mutations confer modest to high-level maturation inhibitor resistance. In most cases, resistance was not associated with a detectable increase in the kinetics of CA-SP1 processing. These results identify mutations that confer resistance to second-generation maturation inhibitors and provide novel insights into the mechanism of resistance.IMPORTANCE HIV-1 maturation inhibitors are a class of small-molecule compounds that block a late step in the viral protease-mediated processing of the Gag polyprotein precursor, the viral protein responsible for the formation of virus particles. The first-in-class HIV-1 maturation inhibitor bevirimat was highly effective in blocking HIV-1 replication, but its activity was compromised by naturally occurring sequence polymorphisms within Gag. Recently developed bevirimat analogs, referred to as "second-generation" maturation inhibitors, overcome this issue. To understand more about how these second-generation compounds block HIV-1 maturation, here we selected for HIV-1 mutants that are resistant to these compounds. Selections were performed in the context of two different subtypes of HIV-1. We identified a small set of mutations at highly conserved positions within the capsid and spacer peptide 1 domains of Gag that confer resistance. Identification and analysis of these maturation inhibitor-resistant mutants provide insights into the mechanisms of resistance to these compounds.

Project description:Lenacapavir (LEN) is a long-acting, highly potent HIV-1 capsid (CA) inhibitor. The evolution of viral variants under the genetic pressure of LEN identified Q67H, N74D, and Q67H/N74D CA substitutions as the main resistance associated mutations (RAMs). Here, we determined high-resolution structures of CA hexamers containing these RAMs in the absence and presence of LEN. Our findings reveal that the Q67H change induces a conformational switch, which adversely affects the inhibitor binding. In the unliganded protein, the His67 side chain adopts the closed conformation by projecting into the inhibitor binding pocket and thereby creating steric hindrance with respect to LEN. Upon the inhibitor binding, the His67 side chain repositions to the open conformation that closely resembles the Gln67 side chain in the WT protein. We propose that the switch from the closed conformation to the open conformation, which is needed to accommodate LEN, accounts for the reduced inhibitor potency with respect to the Q67H CA variant. The N74D CA change results in the loss of a direct hydrogen bond and in induced electrostatic repulsions between CA and LEN. The double Q67H/N74D substitutions exhibited cumulative effects of respective single amino acid changes. An examination of LEN binding kinetics to CA hexamers revealed that Q67H and N74D CA changes adversely influenced the inhibitor binding affinity (KD) by primarily affecting the dissociation rate constant (koff). We used these structural and mechanistic findings to rationally modify LEN. The resulting analog exhibited increased potency against the Q67H/N74D viral variant. Thus, our studies provide a means for the development of second-generation inhibitors with enhanced barriers to resistance. IMPORTANCE LEN is an investigational long-acting agent for future HIV-1 treatment regimens. While ongoing clinical trials have highlighted a largely beneficial profile of LEN for the treatment of HIV-1 infected people with limited therapy options, one notable shortcoming is a relatively low barrier of viral resistance to the inhibitor. Cell culture-based viral breakthrough assays identified N74D, Q67H, and N74D/Q67H capsid changes as the main resistance associated mutations (RAMs). N74D and Q67H capsid substitutions have also emerged in clinical trials in some patients who received subcutaneous LEN. Understanding the structural basis behind viral resistance to LEN is expected to aid in the rational development of improved inhibitors with enhanced barriers to resistance. Here, we report high resolution structures of the main drug resistant capsid variants, which provide mechanistic insight into the viral resistance to LEN. We used these findings to develop an improved inhibitor, which exhibited enhanced activity against the viral Q67H/N74D capsid phenotype compared with that of parental LEN.

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Structural basis of second-generation HIV integrase inhibitor action and viral resistance.

Publications

Structural basis of second-generation HIV integrase inhibitor action and viral resistance.

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