(P27) Molecular Mechanism of N155H/E92Q Resistance to HIV-1 Integrase Inhibitors

Författare/Medförfattare

Saccon E [1], Neogi U [1], Sönnerborg A [1,2,3], Rohit Rao R [3,4], Hill KJ [3,4], Rogers LC [3,4], Cannon J [3,4], Singh K [1,3,4] Presenting author: Saccon E, elisa.saccon@ki.se

Affiliates

Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden [1], Division of Infectious Diseases, Department of Medicine Huddinge, Karolinska Institute, Stockholm, Sweden [2], Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65211, USA [3], Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA [4]

Abstract

Background: HIV-1 integrase resistance mutation E92Q alone or in the background of N155H has been reported for all approved HIV-1 integrase (HIV-IN) strand transfer inhibitors (INSTIs). However, the molecular mechanism of these resistance mutations is not well understood, partly due to the unavailability of a high-resolution structures of HIV-1 IN in complex with the DNA and INSTIs.

Methods: We used a combination of structural, computational and biochemical tools to understand the impact of mutations E92Q, N155H and the double mutation W92Q/N155H on the binding affinity of DNA and dolutegravir (DTG). Induced-fit docking, hybrid quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulations were used to sample the lowest energy conformations of IN/DNA/DTG complexes. Microscale thermophoresis (MST), and gel-based strand transfer assays were used to determine DNA (Kd.DNA) and DTG-binding affinities (Kd.DTG), respectively for the WT, E92Q, N155H and E92Q/N155H integrase derived from HXB2 strain.

Results: The MST assays showed that mutations E92Q and N155H reduced the DNA binding of IN by 2.5- and 3.5-fold, respectively (p≤0.01). However, the Kd.DNA of E92Q/N155H (35±2 nM) was comparable to that of WT (35±2 nM) implying that the addition of E92Q to N155H restored DNA binding affinity of HIV-1 IN. The decreased DNA binding affinity of individual mutations was reflected in the observed flexibility of 3’-end nucleotide of viral DNA in the MD simulations. Increased Kd.DNA of individual mutations was also reflected in decreased 3’-end processing and strand-transfer activities of HIV-1 IN. The DTG binding affinity (Kd.DTG) determined in gel-based assays showed that WT had greatest DTG-binding affinity (lowest Kd.DTG = 21 ± 4 nM) followed by E92Q>N155H>E92Q/N155H. Since the distribution of water molecules near the active site is crucial for catalysis and INSTI binding, we determined the turnover of water molecules near two catalytic metals (A and B) in the presence DNA and DTG. Our results showed that E92Q mutation reduced the visitation of water molecules at both metal ions but N155H mutation increased number of water molecules only at metal B. Estimation of DTG binding energy (ΔG) with IN/DNA complex showed that mutations E92Q, N155H and E92Q/N155H had increased ΔG suggesting that the mutations destabilize INSTI binding to IN.

Conclusion: Based on our results that addition of E92Q in the background of N155H restored DNA binding affinity, we conclude that E92Q is most likely selected to restore the fitness of the virus. Individual mutations alter the visitation of waters required for catalysis. Furthermore, E92Q contributes in destabilizing IN155H/DNA/INSTI complex.