Mass Spectrometric Characterization of HIV-1 Reverse Transcriptase Interactions with Non-nucleoside Reverse Transcriptase Inhibitors

Thammaporn, Ratsupa; Ishii, Kentaro; Yagi-Utsumi, Maho; Uchiyama, Susumu; Hannongbua, Supa; Kato, Koichi

doi:10.1248/bpb.b15-00880

Cited by 5 publications

(4 citation statements)

References 32 publications

(30 reference statements)

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“…For NVP binding, the decrease in signal intensity of the upfield free resonance (−61.9 ppm) is concomitant with an increase in the intensity of the downfield NVP‐bound resonance (−59.7 ppm) (Figure A). A similar observation holds for RPV binding, although the shift occurs at lower NNRTI/protein ratios, consistent with the higher affinity of RPV to RT compared to NVP (Figure B). Assuming that dimeric p66 tfmF /p66 tfmF is in equilibrium between NNRTI‐free and bound forms, with two resonances essentially reporting on the binding, NVP and RPV dissociation constants, K D , were estimated to be 32.…”

Section: Resultssupporting

confidence: 78%

The HIV‐1 p66 homodimeric RT exhibits different conformations in the binding‐competent and ‐incompetent NNRTI site

et al. 2017

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Non-nucleoside inhibitors of HIV-1 reverse transcriptase (RT), NNRTIs, which bind to the p66/p51 heterodimeric RT, also interact with the p66/p66 homodimer, whose structure is unknown. 19F NMR of a single 4-trifluoromethylphenylalanine (tfmF) residue, incorporated into the NNRTI binding pocket of the p66/p66 homodimer at position 181, was used to investigate NNRTI binding. In the NNRTI-bound homodimer complex, two different 19F signals are observed, with the resonance frequencies matching those of the NNRTI-bound p66/p51 heterodimer spectra, in which the individual p66- or p51-subunits were labeled with tfmF at positions 181. These data suggest that the NNRTI-bound p66/p66 homodimer conformation, particularly around residue 181, is very similar to that in the p66/p51 heterodimer, explaining why NNRTI binding to p66/p66 enhances dimer formation.

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Section: Resultssupporting

confidence: 78%

The HIV‐1 p66 homodimeric RT exhibits different conformations in the binding‐competent and ‐incompetent NNRTI site

et al. 2017

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“…The 1:1 complex of FKBP:FK506 was observed in 10 mM ammonium acetate at pH 7.5. The authors observed the protein–ligand interactions by electrospray ionization (ESI) MS. Katta and Chait reported the observation of the hemoglobin complex [ 6 ], and we recently reported three cases of protein–ligand interactions: nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ)–endocrine disruptors [ 7 ], protein tyrosine phosphatase PTPRZ–inhibitor [ 8 ], and HIV-1 reverse transcriptase–drugs [ 9 ]. Typically, the MS study of protein–ligand complexes requires the modification of various components and conditions within a mass spectrometer.…”

Section: Early Ms Studies Of Protein–ligand Interactionsmentioning

confidence: 99%

“…They compared the affinities of estrogen, the compounds, an endocrine disrupter, and a phyto-hormone for the estrogen receptor using MS competition experiments and showed that the rank order of the estimated affinities are consistent with their reported K D values. We recently examined the interaction of the human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) with drugs in different generations by MS under non-denaturing conditions [ 9 ]. The half-concentrations of the drugs where the peak intensities of the HIV-1 RT-drug complexes were equal to those of free HIV-1 RT were well-correlated with the K D values reported in the literature ( Fig.…”

Section: Drug Screening Using Msmentioning

confidence: 99%

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Mass spectrometric analysis of protein–ligand interactions

2016

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The interactions of small molecules with proteins (protein–ligand interactions) mediate various biological phenomena including signal transduction and protein transcription and translation. Synthetic compounds such as drugs can also bind to target proteins, leading to the inhibition of protein–ligand interactions. These interactions typically accompany association–dissociation equilibrium according to the free energy difference between free and bound states; therefore, the quantitative biophysical analysis of the interactions, which uncovers the stoichiometry and dissociation constant, is important for understanding biological reactions as well as for rational drug development. Mass spectrometry (MS) has been used to determine the precise molecular masses of molecules. Recent advancements in MS enable us to determine the molecular masses of protein–ligand complexes without disrupting the non-covalent interactions through the gentle desolvation of the complexes by increasing the vacuum pressure of a chamber in a mass spectrometer. This method is called MS under non-denaturing conditions or native MS and allows the unambiguous determination of protein–ligand interactions. Under a few assumptions, MS has also been applied to determine the dissociation constants for protein–ligand interactions. The structural information of a protein–ligand interaction, such as the location of the interaction and conformational change in a protein, can also be analyzed using hydrogen/deuterium exchange MS. In this paper, we briefly describe the history, principle, and recent applications of MS for the study of protein–ligand interactions.

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Investigation of interactions between cytochrome c and ginsenosides by native mass spectrometry and molecular docking simulations

Zhao

Xing

et al. 2020

Rapid Comm Mass Spectrometry

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RationaleGinsenosides are considered to be the main functional components in ginseng and possess various important pharmacological activities. The study of the interactions between ginsenosides and proteins is indispensable for understanding the pharmacological activities of ginsenosides. In this work, the interactions of ginsenosides with cytochrome c (cyt c) were investigated by native mass spectrometry and molecular docking simulations.MethodsThe interactions of four ginsenosides (Rb1, Rb3, Rf, Rg1) and cyt c in NH4OAc solution were investigated by electrospray ionization linear ion trap mass spectrometry (ESI‐LTQ‐MS). Molecular docking simulations of cyt c complexes were carried out by AutoDock.ResultsThe native mass spectrometry results showed that the four ginsenosides were directly bound to cyt c, with stoichiometric ratios of 1:1 and 2:1 in NH4OAc. The order of relative binding abilities of ginsenosides to cyt c obtained by ESI‐MS was Rb1 > Rb3 > Rf > Rg1, which was consistent with the docking results. Moreover, molecular docking simulations also indicated potential binding sites of cyt c and ginsenosides. Hydrogen‐bond interaction played a very important role in cyt c binding with ginsenosides.ConclusionsIt has been demonstrated that native MS is a useful tool to investigate the interactions of ginsenosides with cyt c. Molecular docking is a good complement to ESI analysis, and can provide information on potential binding sites of cyt c–ginsenoside complexess. This strategy will be helpful to further understand the interactions of proteins and small molecules.

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Mass Spectrometric Characterization of HIV-1 Reverse Transcriptase Interactions with Non-nucleoside Reverse Transcriptase Inhibitors

Cited by 5 publications

References 32 publications

The HIV‐1 p66 homodimeric RT exhibits different conformations in the binding‐competent and ‐incompetent NNRTI site

The HIV‐1 p66 homodimeric RT exhibits different conformations in the binding‐competent and ‐incompetent NNRTI site

Mass spectrometric analysis of protein–ligand interactions

Investigation of interactions between cytochrome c and ginsenosides by native mass spectrometry and molecular docking simulations

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