NMR characterization of HIV-1 reverse transcriptase binding to various non-nucleoside reverse transcriptase inhibitors with different activities

Abstract

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is an important target for antiviral therapy against acquired immunodeficiency syndrome. However, the efficiency of available drugs is impaired most typically by drug-resistance mutations in this enzyme. In this study, we applied a nuclear magnetic resonance (NMR) spectroscopic technique to the characterization of the binding of HIV-1 RT to various non-nucleoside reverse transcriptase inhibitors (NNRTIs) with different activities, i.e., nevirapine, delavirdine, efavirenz, dapivirine, etravirine, and rilpivirine. 1H-13C heteronuclear single-quantum coherence (HSQC) spectral data of HIV-1 RT, in which the methionine methyl groups of the p66 subunit were selectively labeled with 13C, were collected in the presence and absence of these NNRTIs. We found that the methyl 13C chemical shifts of the M230 resonance of HIV-1 RT bound to these drugs exhibited a high correlation with their anti-HIV-1 RT activities. This methionine residue is located in proximity to the NNRTI-binding pocket but not directly involved in drug interactions and serves as a conformational probe, indicating that the open conformation of HIV-1 RT was more populated with NNRTIs with higher inhibitory activities. Thus, the NMR approach offers a useful tool to screen for novel NNRTIs in developing anti-HIV drugs.

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

“…31,32) described. 14) Briefly, the heterodimeric HIV-1 RT protein was purified from cell lysates with sequential use of a diethylaminoethyl (DEAE) cellulose column (Whatman), phosphocellulose P11 column (Whatman), Chelating Sepharose Fast Flow (GE Healthcare) charged with nickel sulfate, and RESOURCE S column (GE Healthcare). Finally, the HIV-1 RT protein was purified by a Superdex-200 (HiLoad 16/60) gel filtration using an FPLC column (GE Healthcare).…”

“…To characterize the interaction of HIV-1 RT with NNRTIs, various biophysical approaches have been applied, including X-ray crystallography, 8,[10][11][12][13] NMR spectroscopy, [14][15][16][17] isothermal titration calorimetry (ITC), [18][19][20][21] and surface plasmon resonance (SPR), 20,22) as well as computational methods. 13,23,24) However, these techniques have often been hampered by the extremely low water solubility of NNRTIs, especially the current most promising diarylpyrimidine-based inhibitors, 25) such as rilpivirine and etravirine.…”

Challenges and approaches in the discovery of human immunodeficiency virus type‐1 non‐nucleoside reverse transcriptase inhibitors

“…After this finding, several authors demonstrated this kind of allosteric modulation of the conformational landscape by NMR experiments . Thammaporn et al characterized the interaction of several NNRTIs with different inhibitory activities. 13 C‐chemical shift of M230 reflects drug‐induced population shifts of the open conformation, where the methyl group of the methionine is more exposed to the solvent, as opposed to the apo closed conformation where the residue is buried in the protein and the peak is not detected because of its low mobility.…”

“…In one simulation, the RT bound to EFV was captured in a closed conformation similar to the apo enzyme, suggesting that the constraint of thumb motion is not complete, so a distribution across open and closed states must exist in the drug‐RT complex . After this finding, several authors demonstrated this kind of allosteric modulation of the conformational landscape by NMR experiments . Thammaporn et al characterized the interaction of several NNRTIs with different inhibitory activities.…”

Application of Site‐Specific Spin Labeling for NMR Detecting Inhibitor‐Induced Conformational Change of HIV‐1 Reverse Transcriptase

“…[9] Various biophysical techniques have revealed that this subunitp rimarily exhibits ac losed conformation in the apo form of HIV-1 RT, whereas non-nucleoside reverset ransferase inhibitors (NNRTIs) bind ah ydrophobic pocket proximal to the polymerase active site, thereby stabilizing itsopen conformation (Figure 1). [10,11] To monitort he inhibitor-induced conformational change of the p66 subunit by using the paramagnetism-assisted NMR technique, we attempted to attach as pin-label group to an intrinsic cysteine residuei nt he p66 subunit. The p51 and p66 subunits share two conserved cysteiner esidues at positions 38 and 280.…”