610 Crystal Structures of HIV-1 Reverse Transcriptase with 4'-ethynyl-2'-deoxyfuranosyl-2,6-diaminopurine Incorporated into the Template-primer Stefan G. Sarafianos*1, Arthur D. Clark Jr.1, Gang Wang2, Barbara L. Gaffney2, Roger A. Jones2, Paul L. Boyer3, Stephen H. Hughes3, Ei-Ichi Kodama4, Hiroaki Mitsuya5, Eddy Arnold1 1Ctr for Advanced Biotechnology and Med and Rutgers Univ, Piscataway, NJ; 2Rutgers Univ, Piscataway, NJ; 3HIV Drug Resistance Prgm, NCI, NIH, Frederick, MD; 4Kyoto Univ, Japan; and 5Kumamoto Univ, Kumamoto, Japan, and NCI-NIH, Bethesda, MD
Background: 4'-ethynyl(4-'E)-2'-deoxynucleoside analogs are potent inhibitors of human immunodeficiency virus in vitro. One of the most potent inhibitors is 4'-E-2'-deoxyribofuranosyl-2,6-diaminopurine (4'-E DAP), which blocks HIV-1 replication in vitro at low concentrations (EC50 = 300 pM) and suppresses the replication of drug-resistant HIV-1 (M41L/T215Y, K65R, L74V, M184V, M41L/T69S-S-G/T215Y, A62V/V75I/F77L/F116Y/Q151M, and Y181C).
Method: To better understand the efficacy of 4'-E DAP against wild-type and drug-resistant RTs, we solved the crystal structures of wild-type RT in complex with double stranded DNA that has 4'-E DAP monophosphate incorporated at the 3' end. The nucleic acid is covalently trapped with 4'-E DAP monophosphate bound at the dNTP-binding site (N complex) (2.9 Å, Rfree = 30.9, R = 27.8).
Results: The electron density for 4'-E DAP monophosphate shows extensive interactions of the 4' ethynyl group with residues of the RT polymerase site, including D185 and A114. The 2-amino group of the diaminopurine of 4'-E DAP interacts with the main chain amino group of G152. The structure was compared with the structure of the N complex of RT with AZTMP-terminated nucleic acid. Preliminary analysis shows structural differences at the N site of the two complexes, including changes in the binding of the 3' terminated primer ends.
Conclusions: These differences may contribute to the favorable resistance profile of 4'-E DAP. We also present structural evidence that an additional nucleotide can be incorporated, extending the 3' end of the chain beyond the 4'-E DAP monophosphate, providing possible insights into the mechanism of inhibition of wild-type RT by 4'-E DAP (and its efficacy against drug-resistant RTs).
