O3. Structural Basis of Inhibition and Resistance Mechanism to EFdA, a highly potent NRTI

Stefan G. Sarafianos1,2,3
Affiliates: 1Bond Life Sciences Center, University of Missouri, Columbia, MO; 2Department of Biochemistry, University of Missouri, Columbia, MO; 3Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO.

Unlike any current clinical nucleoside reverse transcriptase inhibitors (NRTIs), 4’-ethynyl-2-fluoro-2’-deoxyadenosine (EFdA) retains a 3’-OH. EFdA has exceptional potency both in vivo and in vitro, highly efficient against drug-resistant strains and outstanding specificity index. EFdA can act as an immediate or as a delayed chain terminator, by affecting translocation of RT after its incorporation in the nascent DNA chain. It can also be efficiently misincorporated by RT, leading to mismatches that are hard to extend and also protected from excision. The M184V mutation causes modest resistance to EFdA (7.5 fold). To unravel the structural basis of EFdA’s extraordinary activity, unique set of inhibition mechanisms and excellent resistance profile, we have solved crystal structures of wild-type and M184V resistant HIV-1 RT in complex with DNA and EFdA-triphosphate (TP) or with DNA that has EFdA incorporated in it.

Dideoxy-terminated DNA containing a thioalkyl tethered guanosine was covalently cross-linked to RT, mixed with EFdA-TP and crystallized to obtain RT/DNAdd/EFdA-TP complexes. RT-terminated with EFdA at the primer terminus (RT/DNAEFdA) was also crystallized. Crystals were dehydrated, flash-cooled in liquid N2, and diffraction data were collected at Advanced Light Source beamline 4.2.2.

The ternary complex structure of wild-type RT/DNAdd/EFdA-TP was solved at 2.4 Å resolution. This structure reveals the atomic interactions of EFdA-TP that make it a tight binder at the pre-translocation site, thus preventing RT translocation and explaining the inhibition mechanism as a translocation-defective RT inhibitor. Comparison of the 2.9 Å RTM184V/DNAdd/EFdA-TP structure with control structures RTM184V/DNAdd/dA-TP or RTM184V/DNAdd/4’-Ethyl-dA-TP (2.9 Å and 3.2 Å) explain how this mutation affects molecular interactions with EFdA and influences its potency, enhancing our understanding of why resistance to EFdA is difficult to develop. A 2.8 Å structure of an RT/DNA complex with an EFdA-MP incorporated at the 3’ primer end and further extended by a mismatched second EFdA-MP (RT/DNAEFdA-MP –EFdA-MP)also provides insights into the structural details of EFdA in the translocation process as well as mismatched incorporation.

The 4’-ethynyl group of EFdA-TP or EFdA-MP-terminated DNA can bind tightly at a polymerase active site hydrophobic pocket, suppressing translocation and inhibiting further DNA synthesis. The M184V mutation mildly decreases EFdA-TP binding through interactions with the 4’E.