title: Biochemical mechanism of HIV-1 resistance to rilpivirine.
abstract: Rilpivirine (RPV) is a second generation nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) that efficiently inhibits HIV-1 resistant to first generation NNRTIs. Virological failure during therapy with RPV and emtricitabine is associated with the appearance of E138K and M184I mutations in RT. Here we investigate the biochemical mechanism of RT inhibition and resistance to RPV. We used two transient kinetics approaches (quench-flow and stopped-flow) to determine how subunit-specific mutations in RT p66 or p51 affect association and dissociation of RPV to RT as well as their impact on binding of dNTP and DNA and the catalytic incorporation of nucleotide. We compared WT with four subunit-specific RT mutants, p66(M184I)/p51(WT), p66(E138K)/p51(E138K), p66(E138K/M184I)/p51(E138K), and p66(M184I)/p51(E138K). Ile-184 in p66 (p66(184I)) decreased the catalytic efficiency of RT (k(pol)/K(d)(.dNTP)), primarily through a decrease in dNTP binding (K(d)(.dNTP)). Lys-138 either in both subunits or in p51 alone abrogated the negative effect of p66(184I) by restoring dNTP binding. Furthermore, p51(138K) reduced RPV susceptibility by altering the ratio of RPV dissociation to RPV association, resulting in a net reduction in RPV equilibrium binding affinity (K(d)(.RPV) = k(off.RPV)/k(on.RPV)). Quantum mechanics/molecular mechanics hybrid molecular modeling revealed that p51(E138K) affects access to the RPV binding site by disrupting the salt bridge between p51(E138) and p66(K101). p66(184I) caused repositioning of the Tyr-183 active site residue and decreased the efficiency of RT, whereas the addition of p51(138K) restored Tyr-183 to a WT-like conformation, thus abrogating the Ile-184-induced functional defects.
authors.affiliation: Christopher Bond Life Sciences Center, University of Missouri School of Medicine, Columbia, Missouri 65211, USA.
authors.forename: Kamalendra
authors.initials: K
authors.lastname: Singh
authors.forename: Bruno
authors.initials: B
authors.lastname: Marchand
authors.forename: Devendra K
authors.initials: DK
authors.lastname: Rai
authors.forename: Bechan
authors.initials: B
authors.lastname: Sharma
authors.forename: Eleftherios
authors.initials: E
authors.lastname: Michailidis
authors.forename: Emily M
authors.initials: EM
authors.lastname: Ryan
authors.forename: Kayla B
authors.initials: KB
authors.lastname: Matzek
authors.forename: Maxwell D
authors.initials: MD
authors.lastname: Leslie
authors.forename: Ariel N
authors.initials: AN
authors.lastname: Hagedorn
authors.forename: Zhe
authors.initials: Z
authors.lastname: Li
authors.forename: Pieter R
authors.initials: PR
authors.lastname: Norden
authors.forename: Atsuko
authors.initials: A
authors.lastname: Hachiya
authors.forename: Michael A
authors.initials: MA
authors.lastname: Parniak
authors.forename: Hong-Tao
authors.initials: HT
authors.lastname: Xu
authors.forename: Mark A
authors.initials: MA
authors.lastname: Wainberg
authors.forename: Stefan G
authors.initials: SG
authors.lastname: Sarafianos
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009570:Nitriles / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011743:Pyrimidines / Q000737:chemistry / Q000494:pharmacology*; D000068696:Rilpivirine
chemical_list: D009570:Nitriles; D011743:Pyrimidines; D054303:HIV Reverse Transcriptase; D000068696:Rilpivirine