Structural insights into the recognition of nucleoside reverse transcriptase inhibitors by HIV‐1 reverse transcriptase: First crystal structures with reverse transcriptase and the active triphosphate forms of lamivudine and emtricitabine

Abstract

The retrovirus HIV‐1 has been a major health issue since its discovery in the early 80s. In 2017, over 37 million people were infected with HIV‐1, of which 1.8 million were new infections that year. Currently, the most successful treatment regimen is the highly active antiretroviral therapy (HAART), which consists of a combination of three to four of the current 26 FDA‐approved HIV‐1 drugs. Half of these drugs target the reverse transcriptase (RT) enzyme that is essential for viral replication. One class of RT inhibitors is nucleoside reverse transcriptase inhibitors (NRTIs), a crucial component of the HAART. Once incorporated into DNA, NRTIs function as a chain terminator to stop viral DNA replication. Unfortunately, treatment with NRTIs is sometimes linked to toxicity caused by off‐target side effects. NRTIs may also target the replicative human mitochondrial DNA polymerase (Pol γ), causing long‐term severe drug toxicity. The goal of this work is to understand the discrimination mechanism of different NRTI analogues by RT. Crystal structures and kinetic experiments are essential for the rational design of new molecules that are able to bind selectively to RT and not Pol γ. Structural comparison of NRTI‐binding modes with both RT and Pol γ enzymes highlights key amino acids that are responsible for the difference in affinity of these drugs to their targets. Therefore, the long‐term goal of this research is to develop safer, next generation therapeutics that can overcome off‐target toxicity.

Structural features in common of HBV and HIV-1 resistance against chirally-distinct nucleoside analogues entecavir and lamivudine

“…3 and 4). Recently, Bertoletti et al have reported a crystal structure for wild-type HIV-1 RT with bound 3TC-TP (PDB code, 6OUN), which also revealed a unique binding conformation of 3TC-TP, roughly similar to our structure 34 , although with two significant structural differences. First, planar base-pairing does not occur for dG705:3TC-TP in the structure of 6OUN, and the angle of tilt was approximately 22° ( Supplementary Fig.…”

“…It is also noteworthy that in a recently reported experimental structure for the RT WT :DNA:3TC-TP complex, the orientation of oxathiolane to the Met184 was not observed, and the side-chain of Met184 retained normal conformation with a 3.8 Å distance between the sulfur atom of 3TC-TP and Cγ (Supplementary Table S2 and Fig. S5c) 34 . As described above, the RT WT :DNA:3TC-TP complex structure revealed slightly skewed and loose binding of 3TC-TP with Met184 in normal conformation.…”

Post-Catalytic Complexes with Emtricitabine or Stavudine and HIV-1 Reverse Transcriptase Reveal New Mechanistic Insights for Nucleotide Incorporation and Drug Resistance

“…As previously described, the lack of a 3′-OH on the chain-terminated DNA primer results in a lower coordination number of the Mg +2 ion A to the complex (the Mg +2 ion corresponds to metal A in the polymerization mechanism). The lower coordination number results in a weaker binding of the Mg +2 ion A, leading to undetectable electron density [ 12 , 28 ]. The three phosphate groups are firmly anchored in the N site by forming several hydrogen bonds with the side chains of residues R72, D110, and D185, as well as with the backbone of residues V111, D113, and A114.…”

“…Additionally, structural studies of HIV-1 RT by X-ray crystallography have elucidated the mechanism of reverse transcription initiation and incorporation of NRTIs. The combination of these studies has provided insight to how drug resistance to individual NRTIs develops [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. Each NRTI acts as competitive inhibitor for the natural dNTP at the same binding site in RT; however, distinct mechanisms for discrimination and drug resistance have been observed.…”

“…The post-catalytic complex of (-)FTC monophosphate ((-)FTC-MP) incorporated into DNA primer of a dsDNA bound to RT, extends our earlier structural findings with the pre-catalytic complex. Together, these studies provide the structural framework for understanding the molecular mechanism of how this viral polymerase discriminates between (-)FTC and natural nucleotides [ 12 ], and the requirement to undergo a conformation change for successful incorporation by RT.…”