Kalyan Das CABM & Rutgers University, NJ, USA Structural Basis for HIV-1 Reverse Transcriptase Drug Resistance to Zidovudine (AZT) and Tenofovir Kalyan Das CABM & Rutgers University, NJ, USA

NRTI - Inhibition and Resistance Nucleoside/nucleotide analog Gets incorporated at the DNA-primer terminus by RT and acts as a chain terminator NRTI Resistance Different RT mutations or sets of mutations emerge in response to different NRTIs A mutant RT has the ability to discriminate the drug from normal nucleotides Discrimination can occur @ Binding Incorporation Excision 1.

AZT-resistance Mutations ATP’ AZTTP AZT-MP gets incorporated RT removes AZT-MP by excision Excision is reverse of polymerization ATP is the primary excision substrate in vivo ATP excises AZT-MP to form AZTppppA Meyer et al. 1998, PNAS 95:1347 Meyer et al. 1999, Mol. Cell 4:35 Boyer et al. 2001. J. Virol. 75:4832 There was no clue of the role of AZT-resistance mutations for almost 10 years till Mayer et al discovered the excision characteristic of RT.

Methods Five crystal structures were determined wt RT/dsDNA/AZTppppA (3.1 Å resolution) AZTr RT/dsDNA/AZTppppA (3.2 Å) AZTr RT/dsDNA terminated with AZTMP at N-site (3.6 Å) AZTr RT/dsDNA terminated with AZTMP at P-site (2.9 Å) apo AZTr RT (2.6 Å) 1.

dNTP Incorporation and AZT-Resistance Mutations T215Y K219Q D67N K70R M41L AZTppppA primer template palm fingers T215Y K219Q D67N K70R M41L dTTP primer template palm fingers dTTP primer template palm fingers primer template palm fingers

Binding of AZTppppA to AZTr RT/dsDNA Complex Primer AppppAZT R72 T215Y K70R K65 T215Y K70E mutation reduces excision whereas, K70R enhances excision. K70R K70R and T215Y are Excision Enhancing Mutations (EEMs)

ATP binds differently to wild-type and EEM RT  T/Y215 K/R70 K65 R72 YMDD    ' DNA primer template AZTppppA (I) AZTppppA (II) ATP binds differently to wild-type and EEM RT The ATP as an excision substrate binds differently to wild-type RT and EEM/TAM RT Y215 R70 Site I Site II ATP’ Wild-type RT does not have high specificity for ATP binding Superposition of wt HIV-1 RT (green ribbon)/AZTppppA (gray) on G5-mutant RT (cyan/gray ribbon)/AZTppppA (yellow) complexes. The mutated amino acid residues R70 and Y215 apparently enhance binding of ATP( the preferred substrate for AZT excision) at the site II. The molecular surface of G5-mutant RT shows both the sites for ATP-binding. The G5-mutant does not alter the conformation of Site I, rather, generates Site II, which has stronger affinity for ATP than Site I. Summary of the interactions of AZTppppA in the wild-type (A) and AZTr (B) HIV-1 RT excision product complexes. Residues 112-115 are not shown; these residues lie on the bottom of the AZTppppA-binding site in both wild-type and AZTr excision product complexes. The main-chain amide nitrogens of these amino acids are positioned to interact with the negatively charged phosphates of AZTppp-, and may help AZTppppA to bind to RT. The mutations create a new ATP-specific binding site

K65R Background K65R is an NRTI resistance mutation in HIV-1 RT: Selected by TDF, ABC, ddI, and occasionally d4T Observed in 2-5% of antiretroviral-experienced patients Low-level resistance to all NRTIs, with the exception of AZT which remains susceptible 1.

K65R Background K65R biochemical functions: Decreases incorporation rate (kpol) of dNTPs and NRTIs Decreases NRTI excision Increases fidelity Decreases viral replication capacity

Structures of K65R RT/dsDNA/TFV-DP (3. 0 Å; R 0. 251; R-free 0 Structures of K65R RT/dsDNA/TFV-DP (3.0 Å; R 0.251; R-free 0.284) K65R RT/dsDNA/dATP (3.3 Å; R 0.254; R-free 0.286) thumb RNase H fingers TFV-DP/ dATP DNA primer DNA template palm p51 p66

dNTP Binding Site primer : template dNTP K65 b3     palm fingers

K65R and R72 form a Molecular Platform Like K65 in wt RT structures, R65 also interacts with the -phosphate. The guanidinium planes of arginines at positions 65 and 72 stack to form a Molecular Platform. R72 is highly conserved; mutations at R72 impair RT polymerization. How does the platform discriminate TFV-DP from dATP? Y115 Q151 R72 R65

Binding of dATP and TFV-DP to K65R RT Q151 Y115  b3 R72 1.7 R65 dATP TFV-DP

dATP and TFV-DP show alternate R65/R72 rotameric conformations Y115 dATP    Y115 R72 N N   N R65  TFV-DP

K65R mutation: Does not significantly alter interaction of residue 65 with dNTP Forms a Molecular Platform with R72 that may work as a “Check Point” Reduces dNTP incorporation Reduces NRTI excision Increases fidelity 3. The platform has alternate rotameric conformations when TFV-DP vs. dATP binds - Causes discrimination of TFV-DP from dATP

K65R and Excision Enhancing Mutations Decreases excision Increases AZT susceptibility K65R and EEMs (TAMs): Antagonistic for mutation development

K65R and M184V M184V is a primary mutation emerges against 3TC and FTC M184V with K65R Increases resistance to ABC and ddI Partial re-sensitization to TFV

dATP/AZT Y115 V184 R72 R65 Y215 ATP’ R70 3TC/FTC resistance site TFV Excision Enhancing Mutations dATP/AZT Y115 V184 AZT- TP R72  Mg2+ ' ATP’ R65 ' Y215 ATP’ ' R70

Conclusions EEMs create a site for binding ATP as excision substrate K70R and T215Y help ATP binding K65R forms a molecular platform that is responsible for selective NRTI resistance, reduced dNTP incorporation, reduced excision and reduced viral fitness The K65R/R72 platform cross-talks with other NRTI resistance mutations With M184V across the substrate ribose ring Negatively with L74V through the templating base Negatively with EEMs (K70R and T215Y)

Acknowledgements Xiongying Tu Rajiv Bandwar NIH funded Rutgers University CABM Xiongying Tu Rajiv Bandwar Arthur D. Clark, Jr. Joseph Bauman Stefan Sarafianos Steve Tuske Eddy Arnold Chemistry Qianwei Han Barbara L. Gaffney Roger A. Jones Gilead Sciences, Inc. Kirsten White Joy Feng Michael Miller HIV DRP Paul L. Boyer Stephen H. Hughes NIH funded