Background
HIV-1 infection requires the integration of viral DNA into host chromatin, a process mediated by the viral enzyme integrase (IN). This step is effectively blocked by IN strand transfer inhibitors (INSTIs), a key class of antiretroviral drugs. However, resistance to even the most potent INSTIs is becoming more of a clinical problem and the underlying mechanisms of resistance are not fully understood.
Methods
Using cryo-electron microscopy (cryo-EM), we solved new high-resolution structures of WT and drug-resistant HIV-1 IN assemblies with Cabotegravir (CAB) bound. We also performed hybrid quantum mechanics molecular mechanics (QM/MM) and high-resolution molecular dynamics-based (MD) simulations and free energy calculations to delineate the mechanism of resistance.
Results
Here, we investigated the resistance mechanism caused by combinations of mutations at positions E138K, G140A/S, and Q148H/K/R of IN, which affect the efficacy of all known INSTIs. We found that viruses harboring the complex IN mutant E138K/G140A/Q148R led to the largest decrease in EC50 compared to WT viruses. Both the available INSTIs, and new compounds currently in pre-clinical trials, are less effective against the complex IN mutant E138K/G140A/Q148R, indicating that, with the increasing therapeutic use of INSTIs, the prevalence of this variant is likely to increase. Notably, Cabotegravir (CAB), which was approved by the US Food and Drug Administration in 2021 as a long-acting injectable, potently inhibits WT IN, but is particularly susceptible to this complex IN mutant. Using cryo-electron microscopy (cryo-EM), we solved new high-resolution structures of WT and drug-resistant HIV-1 IN assemblies with CAB bound. We also performed molecular dynamics-based free energy calculations to delineate the mechanism of resistance. Our study shows that the E138K/G140A/Q148R mutations disrupt the drug’s efficacy by altering the DDE-Mg (D64+D116+E152-Mg) network at the active site of integrase. The δ+ charge on Arg148 forms a salt bridge with D64, E152, and D116. The mutations cause rearrangements in the active site that reduce the binding of CAB and, by extension, other INSTIs.
Conclusions
Our combined virology, structural biology, and molecular dynamics analyses provides a detailed mechanistic understanding of CAB/INSTI resistance, yielding a foundation for the development of next-generation INSTIs capable of overcoming problematic complex resistant IN variants.