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

Abstract

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) have been developed for the treatment of acquired immunodeficiency syndrome. HIV-1 RT binding to NNRTIs has been characterized by various biophysical techniques. However, these techniques are often hampered by the low water solubility of the inhibitors, such as the current promising diarylpyrimidine-based inhibitors rilpivirine and etravirine. Hence, a conventional and rapid method that requires small sample amounts is desirable for studying NNRTIs with low water solubility. Here we successfully applied a recently developed mass spectrometric technique under non-denaturing conditions to characterize the interactions between the heterodimeric HIV-1 RT enzyme and NNRTIs with different inhibitory activities. Our data demonstrate that mass spectrometry serves as a semi-quantitative indicator of NNRTI binding affinity for HIV-1 RT using low and small amounts of samples, offering a new high-throughput screening tool for identifying novel RT inhibitors as anti-HIV drugs.

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is an attractive target for the development of new drugs for the treatment of acquired immunodeficiency syndrome.^1,2) This enzyme is an asymmetric heterodimer consisting of p66 and p51 subunits and plays an important role in viral replication as it converts single-stranded viral RNA into double-stranded DNA prior to integration into the genome of the human host cell.³⁾ The inhibitors of HIV-1 RT can be divided into two main classes: nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs).^4,5) NNRTIs are expected to be able to circumvent the toxic side effects resulting from nucleoside chain termination.⁶⁾ Accordingly, the NNRTI binding pocket of HIV-1 RT is an important target for the development of novel anti-HIV-1 drugs.^7,8) Five NNRTIs (nevirapine, delavirdine, efavirenz, etravirine, and rilpivirine) have been approved by the United States Food and Drug Administration.⁸⁾ However, efficacies of these inhibitors have been impaired by mutations in HIV-1 RT.⁹⁾ Therefore, continuous development of novel NNRTIs capable of inhibiting both wild-type and mutant HIV-1 RT enzymes is required.

To characterize the interaction of HIV-1 RT with NNRTIs, various biophysical approaches have been applied, including X-ray crystallography,^8,10–13) NMR spectroscopy,^14–17) isothermal titration calorimetry (ITC),^18–21) 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. Hence, ITC and SPR are seldom applied to determine HIV-1 RT binding affinities of NNRTIs except for those with higher water solubility, such as nevirapine and efavirenz. Therefore, a conventional and rapid method that requires small sample amounts is desirable for characterizing the interactions between HIV-1 RT and NNRTIs with lower water solubility.

Recently, the use of mass spectrometry (MS) under non-denaturing conditions has become a powerful tool for analyzing non-covalently associated biomacromolecular complexes.^26–28) Several reports have shown that MS can be used to characterize the binding of drugs to target enzymes in terms of stoichiometry, specificity, and stability.^29,30) This method is advantageous because it requires small sample amounts. Because of its rapidity, sensitivity, and ability to directly determine binding stoichiometry, MS is potentially applicable as a superior high-throughput screening method for developing anti-HIV drugs.

In this study, we test the applicability of MS to the characterization of HIV-1 RT binding to four NNRTIs (nevirapine, efavirenz, etravirine, and rilpivirine) with different inhibitory activities (Fig. 1).

Fig. 1. Structures and Molecular Weights of Nevirapine, Efavirenz, Rilpivirine, and Etravirine

RESULTS AND DISCUSSION

First, we assessed the heterodimeric state of HIV-1 RT using MS. The mass spectrum of HIV-1 RT exhibited ion series giving a molecular mass of 117873±29 Da (Fig. 2). Under a denaturing condition with 30% (v/v) formic acid, HIV-1 RT exhibited two ion series corresponding to the molecular masses of the p51 (52632 Da) and p66 (65168 Da) subunits. Thus, we confirmed that the ion series observed under the non-denaturing condition corresponds to the heterodimer composed of the p66 and p51 subunits.

Fig. 2. A) MS Spectra of HIV-1 RT Titrated with Etravirine; B) The Titration Curve of HIV-1 RT-Etravirine Complex Formation against Etravirine Concentration

A) The concentration of HIV-1 RT was fixed at 3.8 µM and that of etravirine was (a) 0 µM, (b) 2 µM, (c) 5 µM, or (d) 10 µM. Filled circles show the ion series of etravirine-bound HIV-1 RT. B) The ratios of etravirine-bound HIV-1 RT to total HIV-1 RT were estimated from the MS peak intensities corresponding to the +19 charge states.

Next, we attempted to characterize the interactions of the p66/p51 heterodimer of HIV-1 RT with NNRTIs. Figure 2 shows the spectral changes of HIV-1 RT upon addition of etravirine. In the MS analysis, intensities of the original ion peaks of the p66/p51 heterodimer were attenuated on titration with etravirine, with concomitant appearance of a new peak series. The molecular mass determined for this complex was 118296±32 Da, which corresponds to a 1 : 1 : 1 complex composed of p66, p51, and etravirine (with a calculated mass of 435.3 Da). Furthermore, these data indicated that 10 µM etravirine was sufficient to form a clearly observable complex with 3.8 µM HIV-1 RT.

Based on these observations, we measured a series of MS spectra of HIV-1 RT in the presence of one of the other compounds at the same concentration. Consequently, a new peak series from the NNRTIs-HIV-1 RT complex was observed upon addition of rilpivirine with similar spectral changes to etravirine, whereas less pronounced and virtually no spectral changes were induced by efavirenz and nevirapine, respectively (Fig. 3). It has been reported that the dissociation constants for the binding of HIV-1 RT to nevirapine, efavirenz, and rilpivirine are 4×10⁻⁷ M,³¹⁾ 17×10⁻⁸ M,³¹⁾ and 18×10⁻⁹ M,³²⁾ respectively. Thus, the affinities of these NNRTIs are in agreement with the MS peak intensities of their complex with HIV-1 RT, indicating that relative affinities of the inhibitors to HIV-1 RT can be estimated by MS measurements (Fig. 4). In addition, our previous NMR analysis revealed that the methyl ¹³C chemical shifts of methionine-230 of HIV-1 RT, which is in close proximity to the NNRTI binding pocket, can be used as an indicator of the efficacy of NNRTI.¹⁴⁾ The NMR data suggest that etravirine and rilpivirine have comparable binding affinity for HIV-1 RT, consistent with the present MS results.

Fig. 3. MS Spectra of HIV-1 RT in the Presence of (a) Etravirine, (b) Rilpivirine, (c) Efavirenz, or (d) Nevirapine

The concentration of HIV-1 RT and each compound were fixed at 3.8 and 10 µM, respectively. The right panel shows the enlarged views of the peaks corresponding to the +19 charge states. Filled circles show the ion series of NNRTI-bound HIV-1 RT.

Fig. 4. Correlation between the Dissociation Constants (K_d) and the Relative Affinities Estimated from MS Titration Data for Inhibitor Binding to HIV-1 RT

The C₅₀ value indicates inhibitor concentration (µM) at which the 50% of total HIV-1 RT forms a complex with each inhibitor. The K_d values of rilpivirine, efavirenz, and nevirapine have been reported in the literatures.^31,32)

In the present study, we successfully applied MS to observe the interactions between HIV-1 RT and various NNRTIs under non-denaturing conditions. Our data demonstrate that MS serves as a semi-quantitative indicator of NNRTI binding affinity for HIV-1 RT using low concentrations and small amounts of samples. Therefore, MS could potentially be used as a new high-throughput screening technique to identify novel RT inhibitors for anti-HIV drug development.

MATERIALS AND METHODS

NNRTIs

Powdered rilpivirine and etravirine were purchased from MedChem Express. Nevirapine and efavirenz were purchased from Sigma. A stock solution (0.2 mM) of each inhibitor was prepared in dimethyl sulfoxide (DMSO).

Preparation of HIV-1 RT Protein

The recombinant plasmids containing the HIV-1 RT genes, pGEX3X³³⁾ for p66 and pET33B³⁴⁾ for p51, were previously constructed. Recombinant HIV-1 RT was expressed and purified as previously 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). All purification steps for HIV-1 RT were performed at 4°C with buffer containing protease inhibitors.

MS Measurements

The purified HIV-1 RT sample was buffer-exchanged to 200 mM ammonium acetate, pH 7.6, by passing through a Bio-Spin 6 column (Bio-Rad). HIV-1 RT (3.8 µM) was incubated with each inhibitor in the presence of 5% DMSO, and the samples were analyzed by nanoflow electrospray using in-house made gold-coated glass capillaries (2–5 µL of sample was loaded for each analysis). Under a denaturing condition, 30% (v/v) formic acid was added to the sample solution. Spectra were recorded using a SYNAPT G2-Si HDMS mass spectrometer (Waters) in positive ionization mode at 1.33 kV with 150 V of sampling cone voltage and source offset voltage, 5 V of trap and 0 V of transfer collision energy, and 5 mL/min trap gas flow. Spectra were calibrated using 1 mg/mL cesium iodide and analyzed using MassLynx (Waters) and IGOR Pro (Wave Metrics) software.

Acknowledgments

We thank Yukiko Isono and Tuanjai Somboon (Institute for Molecular Science) for their help in sample preparation during the early stage of this study. This work was supported in part by grants (25102001, 25102008, 15K21708 and 15H02491 to K.K.; 25121722 and 26102530 to S.U.; 15K21680 to M.Y.-U.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan as well as by the Okazaki ORION project and the Joint Studies Program (2014–2015) in the Okazaki BIO-NEXT project of the Okazaki Institute for Integrative Bioscience. This work was also supported by the Thailand Research Fund (RTA5380010). R.T. is grateful to the Royal Golden Jubilee PhD Program (Grant No. 3. C.KU/51/B.1) for the scholarship, to the Faculty of Science, Kasetsart University (Outbound Research Student Exchange, ORSE) for supporting, and to the Institute for Molecular Science (IMS), Japan, for the research fellowship.

Conflict of Interest

The authors declare no conflict of interest.

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