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Transition states of native and drug-resistant HIV-1 protease are the same.

ABSTRACT: HIV-1 protease is an important target for the treatment of HIV/AIDS. However, drug resistance is a persistent problem and new inhibitors are needed. An approach toward understanding enzyme chemistry, the basis of drug resistance, and the design of powerful inhibitors is to establish the structure of enzymatic transition states. Enzymatic transition structures can be established by matching experimental kinetic isotope effects (KIEs) with theoretical predictions. However, the HIV-1 protease transition state has not been previously resolved using these methods. We have measured primary (14)C and (15)N KIEs and secondary (3)H and (18)O KIEs for native and multidrug-resistant HIV-1 protease (I84V). We observed (14)C KIEs ((14)V/K) of 1.029 ± 0.003 and 1.025 ± 0.005, (15)N KIEs ((15)V/K) of 0.987 ± 0.004 and 0.989 ± 0.003, (18)O KIEs ((18)V/K) of 0.999 ± 0.003 and 0.993 ± 0.003, and (3)H KIEs ((3)V/K) KIEs of 0.968 ± 0.001 and 0.976 ± 0.001 for the native and I84V enzyme, respectively. The chemical reaction involves nucleophilic water attack at the carbonyl carbon, proton transfer to the amide nitrogen leaving group, and C-N bond cleavage. A transition structure consistent with the KIE values involves proton transfer from the active site Asp-125 (1.32 ?) with partial hydrogen bond formation to the accepting nitrogen (1.20 ?) and partial bond loss from the carbonyl carbon to the amide leaving group (1.52 ?). The KIEs measured for the native and I84V enzyme indicate nearly identical transition states, implying that a true transition-state analogue should be effective against both enzymes.

SUBMITTER: Kipp DR

PROVIDER: S-EPMC3340095 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Transition states of native and drug-resistant HIV-1 protease are the same.

Kipp D Randal DR Hirschi Jennifer S JS Wakata Aya A Goldstein Harris H Schramm Vern L VL

Proceedings of the National Academy of Sciences of the United States of America 20120409 17

HIV-1 protease is an important target for the treatment of HIV/AIDS. However, drug resistance is a persistent problem and new inhibitors are needed. An approach toward understanding enzyme chemistry, the basis of drug resistance, and the design of powerful inhibitors is to establish the structure of enzymatic transition states. Enzymatic transition structures can be established by matching experimental kinetic isotope effects (KIEs) with theoretical predictions. However, the HIV-1 protease trans ...[more]

PMID: 22493227

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Project description:Drug resistance is a serious problem for controlling the HIV/AIDS pandemic. Current antiviral drugs show several orders of magnitude worse inhibition of highly resistant clinical variant PRS17 of HIV-1 protease compared with wild-type protease. We have analyzed the effects of a common resistance mutation G48V in the flexible flaps of the protease by assessing the revertant PRS17V48G for changes in enzyme kinetics, inhibition, structure, and dynamics. Both PRS17 and the revertant showed about 10-fold poorer catalytic efficiency than wild-type enzyme (0.55 and 0.39 μM-1min-1 compared to 6.3 μM-1min-1). Clinical inhibitors, amprenavir and darunavir, showed 2-fold and 8-fold better inhibition, respectively, of the revertant than of PRS17, although the inhibition constants for PRS17V48G were still 25 to 1,200-fold worse than for wild-type protease. Crystal structures of inhibitor-free revertant and amprenavir complexes with revertant and PRS17 were solved at 1.3-1.5 Å resolution. The amprenavir complexes of PRS17V48G and PRS17 showed no significant differences in the interactions with inhibitor, although changes were observed in the conformation of Phe53 and the interactions of the flaps. The inhibitor-free structure of the revertant showed flaps in an open conformation, however, the flap tips do not have the unusual curled conformation seen in inhibitor-free PRS17. Molecular dynamics simulations were run for 1 μs on the two inhibitor-free mutants and wild-type protease. PRS17 exhibited higher conformational fluctuations than the revertant, while the wild-type protease adopted the closed conformation and showed the least variation. The second half of the simulations captured the transition of the flaps of PRS17 from a closed to a semi-open state, whereas the flaps of PRS17V48G tucked into the active site and the wild-type protease retained the closed conformation. These results suggest that mutation G48V contributes to drug resistance by altering the conformational dynamics of the flaps.

Dataset Information

Transition states of native and drug-resistant HIV-1 protease are the same.

Publications

Transition states of native and drug-resistant HIV-1 protease are the same.

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