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HIV-1 Reverse Transcriptase: Drug Resistance Mechanisms. Steps of DNA Polymerization. RT. RT/DNA. RT/DNA/dNTP. fingers. thumb. DNA n. PPi. dNTP. E ´ /DNA n. E ´ /DNA n+1. E ´ /DNA n /dNTP. E. E*/DNA n /dNTP. E + DNA. Conformational change/ catalysis. dNTP binding. DNA binding.
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Steps of DNA Polymerization RT RT/DNA RT/DNA/dNTP fingers thumb DNAn PPi dNTP E´/DNAn E´/DNAn+1 E´/DNAn/dNTP E E*/DNAn/dNTP E + DNA Conformational change/ catalysis dNTP binding DNA binding Translocation Sarafianos et al. (1999) Chem. & Biol. 6, R137-R145
Nucleoside Analogs and Nonnucleoside RT Inhibitors • Nucleoside analogs (NRTIs) that are used to treat HIV-1 infections all lack the normal 3’ OH and act as chain terminators when incorporated into viral DNA • Nonnucleoside RT inhibitors (NNRTIs) bind to HIV-1 RT near, but not at, the polymerase active site distorting the enzyme and blocking the chemical step of viral DNA synthesis
Drug Binding Sites in HIV-1 RT NRTI NNRTI
Sites of Drug Resistance Mutations in HIV-1 RT NNRTI resistance mutation sites NRTI resistance mutation sites
Nonnucleoside RT Resistance Mechanisms • Mutations that reduce the interactions between RT and the bound drug (Y181C, Y188L). • Mutations that cause steric hindrance with the bound drug (G190A/S). • Mutation that makes it more difficult for the NNRTI to enter the binding pocket (K103N). • Indirect effects (L100I, V106A, V108A).
NRTI Resistance • Resistance to nucleoside analogs (NRTIs) implies that the mutant reverse transcriptase (RT) has an enhanced ability to discriminate between the NRTI and the normal dNTPs. • This discrimination can occur if the NRTI is incorporated less efficiently (exclusion) or by enhanced excision of the NRTI after it has been incorporated (ATP-dependent pyrophosphorolysis).
NRTI Resistance Mechanisms • Exclusion: M184V, 3TC/FTC resistance; steric hinderance • Exclusion: K65R, Q151M resistance to multiple NRTIs, altered interactions with the 3’OH of the incoming dNTP • Excision: TAMs (T215F/Y and friends) cause AZT resistance by enhanced ATP binding • Fingers insertions: extend TAMs to cause excision of many NRTIs
AZT Resistance (Excision) • Excision involves pyrophosphorolysis (the reverse of polymerization). • AZT-resistant RTs preferentially excise AZTMP. • AZT-resistance mutations enhance the ability of RT to bind ATP, the in vivo pyrophosphate donor. • AZT is excised easily because the long azido group interferes with translocation: an AZT terminated primer preferentially resides at the N (active) site where it can be excised
Closed Complex Reduces Excision Stable Complex No Excision b3-b4 Loop
AZTMP Excision * * ATP Binding Mutations Unstable Complex Excision AZT MP b3-b4 Loop
Y183 primer template P M184 D186 Y115 N D185 AZTMP D110 g D67N b fingers K219E a D113 T215Y K70R M41L ATP
An AZT Terminated Primer in the P Site Causes Steric Hindrance with a dNTP in the N site P Site (AZTMP) N Site (dNTP) Active Site
AZT Resistance: Q151M • Certain NRTI combination therapies (AZT + ddI + ddC) select Q151M in HIV-1 RT. However, Q151M appears in only 5% of the treated HIV-1 patients. • There is less data (fewer treated patients) but HIV-2 prefers to use the Q151M pathway for AZT resistance • In HIV-1 (but not HIV-2) the Q151Mutation is usually accompanied by additional mutations • Why does HIV-1 predominantly use an ATP-dependent excision pathway (T215Y/F) while HIV-2 predominantly uses an AZT exclusion pathway (Q151M)?
AZTTP Inhibition of Polymerization of HIV-1RT, HIV-2 RT, and Their Q151M Mutants
HIV-1 RT (WT and Mutants) Are Better at AZTMP Excision than HIV-2 RT (WT and Mutants)
ATP Binding sites in HIV-1 RT and HIV-2 RT P P 185 AZTMP N 185 AZTMP N 112 115 ATP 115 215 112 209 113 214 ATP 117 214 116 118 113 116 215 117 41 211 44 5 44 41 4 1 46 46 2 4 3 HIV-1 RT HIV-2 RT
HIV-1 and HIV-2 AZT Resistance • Each virus prefers a resistance pathway that is best suited to extend the properties of their respective wild-type RTs. • Viewed in this light, the ability of HIV-1 to develop AZT (and multi-NRTI) resistance using ATP-mediated excision is an unfortunate coincidence based on the existence of a nascent ATP binding site that appears to have no normal function.
Delayed Chain Terminators • HIV-1 excision works efficiently because the NRTI remains at the end of the primer strand where it can be excised. • Delayed chain terminators block DNA synthesis several nucleotides after they have been incorporated. • As expected, delayed chain terminators block the excision reaction and inhibit the growth of viruses that replicate using excision proficient RTs.
Conclusions • There are a number of distinct mechanisms for NRTI and NNRTI resistance. • Resistance mechanisms evolve as logical extensions of the properties of wild type RT. • Understanding the mechanism(s) of RT resistance makes it possible to develop strategies (and drugs) that counteract these mechanisms .
Acknowledgments NCI-Frederick CABM/Rutgers Paul L. Boyer Stefan Sarafianos* Pat Clark Kalyan Das John Julias Eddy Arnold Victor Marquez Tel Aviv Amnon Hizi
N-MCT Inhibits HIV-1 Viruses that Replicate Using Excision-Proficient RTs in HSV TK+ Cells Relative Infectivity µM North-MCT
HIV Infection/Mortality • Over 3 million deaths in 2003; about 7 per minute • 40-50 million infected worldwide • In 2003, about 5 million new infections, mostly in Africa, South and Southeast Asia • About 1 million already infected in the US; about 45,000 new infections and 15,000 deaths in 2003
HIV Evolution • HIV replication is error prone: Error rate ca. 1 per genome per replication cycle • The rapid replication of the virus, together with the error rate and high viral load causes the virus to evolve rapidly • The rapid evolution of the virus makes the problems of vaccine development and drug therapy particularly difficult
Anti-HIV Drugs and Drug Targets • Target should be essential and conserved; enzymes are better targets (RT, PR, IN) • HIV develops resistance to all drugs • RT has at least two separate drug targets (there may be more); PR and IN have one • Understand resistance; develop drugs that are effective against resistant viruses
Anti-HIV Drugs and Drug Resistance • Drugs do not cure an HIV infection • Drugs need to be taken (regularly) for the life of the patient • For drugs to be effective replication must be blocked completely (stop evolution of resistance) • It takes at least three drugs in combination to fully block viral replication/evolution
Flexible inhibitor Rigid inhibitor Torsional changes (wiggling) Steric hindrance Reorientation and repositioning (jiggling)
Two NRTI Drug Resistance Paradigms: Reduced Incorporation and Enhanced Excision
Clash Between Incorporated AZT and Active Site Aspartic Acid Prevents Translocation
WT Complex N superposed on WT Complex P template primer P site D185 D186 AZTMP D110 N site AZTMP Q151 R72 fingers
P site (priming site) Primer dNTP N site (nucleotide binding site) 3’OH a Catalytic carboxylates b g Released as pyrophosphate
Y183 primer template P M184 D186 Y115 N D185 AZTMP D110 g D67N b fingers K219E D113 T215Y K70R M41L PPi
NRTI Excision * * ATP Binding Mutations Unstable Complex Excision b3-b4 Loop Fingers Mutations
Excision/Extension Assay +ATP, dNTPs, NRTITPs
HIV-1 RT and HIV-2 RT Sequences HIV-1 (BH10) HIV-2 (ROD) ---PGIRYQYNVLPQGWKGSPAIFQ--- || || | |||||||||||||| ---PGKRYIYKVLPQGWKGSPAIFQ--- 151 ---IYQYMDDLYVGSDLEIGQHRTKIEELRQHLLRWGLTTPDKKHQKEPP--— | |||||• ••|| | • |• | | •||| | ||•|| ---IIQYMDDILIASDRTDLEHDRVVLQLKELLNGLGFSTPDEKFQKDPP--- 185215 219
ATP-Mediated AZTMP Excision/Extension Deblock and Extend 100 uM each dNTP vary ATP
PPi-Mediated AZTMP Excision/Extension Deblock and extend 100 uM each dNTP vary PPi
Q151 in HIV-1 and HIV-2 RT 74 115 151 73 HIV-1 /DNA/dNTP HIV-1 116 HIV-2