HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem, and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir (DTG). We present 11 new cryo-EM structures of drug-resistant HIV-1 intasomes bound to DTG or 4d, with better than 3-Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K + G140A/S + Q148H/K/R and show why 4d maintains potency better than DTG. These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.

While drug resistance mutations can often be attributed to the loss of direct or solvent-mediated protein−ligand interactions in the drug-mutant complex, in this study we show that a resistance mutation for the picomolar HIV-1 capsid (CA)-targeting antiviral (GS-6207) is mainly due to the free energy cost of the drug-induced protein side chain reorganization in the mutant protein. Among several mutations, M66I causes the most suppression of the GS-6207 antiviral activity (up to ~84,000-fold), and only 83- and 68-fold reductions for PF74 and ZW-1261, respectively. To understand the molecular basis of this drug resistance, we conducted molecular dynamics free energy simulations to study the structures, energetics, and conformational free energy landscapes involved in the inhibitors binding at the interface of two CA monomers. To minimize the protein−ligand steric clash, the I66 side chain in the M66I−GS-6207 complex switches to a higher free energy conformation from the one adopted in the apo M66I. In contrast, the binding of GS-6207 to the wild-type CA does not lead to any significant M66 conformational change. Based on an analysis that decomposes the absolute binding free energy into contributions from two receptor conformational states, it appears that it is the free energy cost of side chain reorganization rather than the reduced protein−ligand interaction that is largely responsible for the drug resistance against GS-6207.

HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir ( DTG ). We present 11 new cryo-EM structures of drug resistant HIV-1 intasomes bound to DTG or 4d , with better than 3 Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K+G140A/S+Q148H/K/R and show why 4d maintains potency better than DTG . These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.

Predicting the positions of water molecules at the protein interface remains a formidable challenge in structural biology, fueling active research in this field. Here, we present a novel approach based on molecular dynamics (MD) simulations that utilize statistical thermodynamic signatures of water at protein interfaces that can be used to improve the accuracy of water placement in maps derived by cryogenic electron microscopy (cryo-EM). We employ an analysis based on the excess chemical potential, or the Work to Transfer (WT) a water molecule from the bulk to the interface. WT is a measure of the thermodynamic balance between the interaction energy of an interfacial water molecule with the protein and its free energy of interaction with all the other solvent molecules. WT is proportional to the log ratio of the local density of water molecules at the protein interface to the bulk density. Using apoferritin as a benchmark system, we found that 85% of the top 100 water locations with the most favorable excess chemical potential values are observed in one or more structures whose locations were determined from high resolution cryo-EM maps deposited in the PDB. 70% of the top 200 water locations indexed by excess chemical potential were also observed in PDB structures derived from the cryo-EM maps. The MD simulations are performed without experimental density restraints, and yet the water positions with favorable WT values correlate strongly to their corresponding position within experimentally defined maps. This work paves the way for the development of a cryo-EM water placement and refinement tool that integrates molecular dynamics simulations of the excess chemical potential with cryo-EM data for accurate modeling of water networks.