A Sensitive Assay Using a Native Protein Substrate For Screening HIV-1 Maturation Inhibitors Targeting the Protease Cleavage Site between Matrix and Capsid

Cell Culture and DNA Transfection

293T cells and the TZM-BL cells (NIH AIDS Research and Reference Reagent Program) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum in the presence of penicillin and streptomycin at 37°C with 5% CO2. 293T cells are derived from human embryonic kidney cells⁵⁰, and TZM-BL cells are HeLa cells that stably express CXCR4, CCR5, and CD4⁵¹. For cotransfection, 0.5 × 10⁶ 293T cells were seeded onto a six-well plate 24 h before transfection. 293T cells were transfected with a total of 4 µg of wild-type and mutant plasmid constructs using FuGENE 6 transfection reagent (Promega).

Infectivity Assay

The culture supernatant containing virus particles was harvested 48 h after transfection and filtered through a 0.45-µm-pore-size membrane (Millipore) to remove cell debris. The culture supernatant was diluted 1:50 or 1:100 and used to infect 2 × 10⁴ TZM-BL cells in a 96-well plate. The TZM-BL indicator cells express the luciferase gene and the lacZ gene under the control of the HIV-1 LTR. For the luciferase assay, infected TZM-BL cells were lysed 48 h postinfection. Briefly, the culture medium was removed from each well, and the cells were washed with phosphate-buffered saline. A 50 µL aliquot of 1× reporter lysis buffer (Promega) was added to the cells, and the cells were kept at −80°C. After one freeze-thaw cycle, 25 µL of cell lysate was transferred into a 96-well assay plate (Costar), and luciferase activity was measured using a luminometer (Promega).

Expression and Purification of HIV-1 PR Substrates

Expression of recombinant proteins in E. coli BL21 (DE3) was carried out by a modification of the established protocols. ^{52, 53}, Briefly, recombinant proteins were expressed in E. coli BL21 (DE3) grown in MagicMedia (Invitrogen) for 7 h and the cells were collected by centrifugation. The cell pellet was lysed in Tris-buffered saline (25 mM Tris base, 3 mM KCl, and 140 mM NaCl) at pH 7.5 with 1 mM dithiothreitol (DTT) and 1% Triton X-100 then sonicated. Following clarification by centrifugation, the recombinant proteins containing a His6-tag at the N-terminus were purified from the soluble fraction by affinity chromatography using a nickel-chelating column (Novagen). The purified recombinant proteins were dialyzed against 20 mM sodium acetate (pH 7.0), 1 mM EDTA, 2 mM DTT, and 10% glycerol.⁵⁴ The p15-C2S protein was purified under denaturing conditions during the nickel-chelating column chromatography (Invitrogen) and refolded by a step-wise dialysis. The dialysis buffers used for refolding contained 5 M urea, 400 mM NaCl, 12.5% glycerol, and 2.5 mM DTT at pH 5.5 for the initial dialysis, 2 M urea, 400 mM NaCl, 12.5% glycerol, and 2.5 mM DTT at pH 6.0 for the second dialysis, and 400 mM NaCl, 12.5% glycerol, and 2.5 mM DTT at pH 6.5 for the last dialysis. Protein concentrations were determined by using the Bradford assay, and the purity of the preparations was analyzed by protein staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).⁵⁵

Protease Expression and Purification

The HIV-1 protease was expressed in E. coli TAP 106 cells and purified from inclusion bodies as previously described.⁵⁶ Briefly, the inclusion body centrifugation pellet was dissolved in 50% acetic acid followed by another round of centrifugation to remove impurities. Size exclusion chromatography was used to separate high molecular weight proteins from the protease. The protein was refolded in 50 mM sodium acetate at pH 5.5, 5% ethylene glycol, 10% glycerol, and 5 mM DTT.

Gel-based HIV-1 Protease Assay using FlAsH-labeled Protein Substrates

The gel-based HIV-1 protease assay was carried out following the protocol described previously.⁵⁷ Briefly, 3 µM of each of two substrates, MA/CA and MA/CAΔ, were labeled with 6 µM FlAsH reagent in a tube with a total volume of 228 µL overnight at room temperature. Proteolysis of the substrates was performed in the proteolysis buffer (50 mM sodium acetate, pH 7.0, 150 mM NaCl, 1 mM EDTA, 2 mM 2-mercaptoethanol, and 10% glycerol) at 30°C by addition of 0.1 µM HIV-1 PR (50 nM dimeric HIV-1 PR based on protein concentration). Aliquots of 24 µL were taken at various time points and the reaction was stopped by adding 4× SDS-PAGE loading buffer for further analysis by SDS-PAGE. An aliquot of 24 µL was taken for the 0 min time point prior to the addition of 50 nM HIV-1 PR. After SDS-PAGE, the gels were briefly rinsed with water and the fluorescently labeled protein bands were visualized by fluorescence imaging using a Typhoon 9400 (GE healthcare) with excitation at 488 nm and emission at 526 nm.

FP Assay in a 96-well Plate

For end-point analysis, the substrate MA/CAΔ was serially diluted 2-fold from 4 µM to 0.25 µM in a 96-well half-area plate (Costar) and 200 nM FlAsH reagent was added to each well for the overnight labeling reaction in a volume of 40 µL. The labeling reaction was performed at room temperature in proteolysis buffer. The well designated as 0 µM substrate contained only 200 nM FlAsH reagent. After the labeling reaction, proteolysis was performed in the absence or presence of 1 µM PR at 30°C for 3 h in a final volume of 50 µL, and then FP values were measured with excitation at 480 nm (30 nm bandpass) and emission at 535 nm (40 nm bandpass) using a PerkinElmer En Vision 2103 Multilabel Reader. For the time course analysis, 200 nM FlAsH reagent was incubated overnight (~15 h) with 2 µM substrate at room temperature in the same proteolysis buffer described above. An aliquot of HIV-1 PR was added to make the PR 50 nM in the reaction volume containing the substrate MA/CAΔ labeled with a FlAsH reagent. The final volume in each well was 50 µL. Immediately after the addition, the reactions were mixed briefly by pipetting and the FP value was recorded every 5 min for 3 h using the same conditions as described for the end-point analysis. For the FP assay in the presence of saquinavir (SQV), 1 nM, 10 nM, 15 nM, 20 nM, or 25 nM SQV was added in DMSO (4% final concentration) to the reaction prior to the addition of the PR. FP values are presented as millipolarization units (mP) in which P can be calculated by a standard equation.⁵⁸

HTS Assay Validation in 384-well Plates

The FP assay was validated in 384-well plates following the procedure described for 96-well plate with modifications. An aliquot of 22 µM MA/CAΔ substrate was labeled with 2 µM FlAsH reagent in 50 mM HEPES buffer at pH 6.8 containing 1 mM DTT at room temperature for 1 h. Proteolysis in a 384-well plate was performed by the addition of 50 nM HIV-1 PR in the presence of 50 mM HEPES at pH 6.8, 150 mM NaCl, 2 mM DTT, 0.01% BSA, and 1% DMSO at 37°C for 2 h. The final concentration of the substrate MA/CAΔ in the proteolysis reaction was 1 µM and the final volume in each well was 30 µL. FP values were measured with excitation at 485 nm and emission at 530 nm using an Aquest GT Multimode Microplate Reader (Molecular Devices). All liquid handling was operated by NanoScreen Robot NS-1536 (NanoScreen). The validation process was carried out on three consecutive days and two plates were assayed on each day. Each assay plate contained two columns of positive and negative control wells containing wild-type HIV-1 PR and heat-inactivated HIV-1 PR, respectively. All raw data were analyzed using ScreenAble (ScreenAble Solution) to determine Z′-value and coefficient of variation (CV) for each plate.

Test Screening with LOPAC¹²⁸⁰ Compound Library

The Library of Pharmacologically Active Compounds (LOPAC) was purchased from Sigma-Aldrich, dissolved in DMSO by the manufacturer. Each compound was stored in DMSO to a concentration of 10 mM in 384-well plates. For the screening, each compound was further diluted to a concentration of 100 µM in the HEPES-based proteolysis buffer. The LOPAC collection was screened in duplicate in 384-well plates. Briefly, 13.5 µL of 2.2 µM MA/CAΔ labeled with FlAsH tag, 13.5 µL of 110 nM HIV-1 PR, and 3 µL of the diluted compound were transferred into the assay plate, and the plate was incubated at 37°C for 3 h before measuring FP values using an Aquest GT Multimode Microplate Reader (Molecular Devices). The final volume in each well was 30 µL and the final concentration for each compound, the HIV-1 PR, and the substrate in each assay reaction was approximately 10 µM, 50 nM, and 1 µM, respectively. All liquid handling was operated by a NanoScreen Robot NS-1536 (NanoScreen). Each assay plate contained two columns of positive and negative control wells, which were run with DMSO alone without a compound. The positive control wells contained wild-type HIV-1 PR and the negative control wells contained heat-inactivated HIV-1 PR. Raw data were analyzed using ScreenAble (ScreenAble Solution) to determine Z′–value, CV, and percent inhibition of each assayed compounds. To calculate the percent inhibition of cleavage, the mean value from the positive control wells was considered as 0% and the mean value from the negative control wells was considered as 100%. The degree of agreement between the two measurements was determined by Bland-Altman analysis.⁵⁹

Design and Expression of MA/CA Substrates

As a first step in searching for novel maturation inhibitors that would selectively block proteolytic processing at the highly sensitive MA/CA site, we have developed a proteolytic cleavage assay based on fluorescence polarization (FP) (Figure 2A) in both 96- and 384-well plates using a series of naturally folded MA/CA protein substrates containing an intact MA/CA cleavage site (Figure 2B). To monitor the cleavage at this site by the HIV-1 PR in either an FP assay or a gel-based assay, a tetracysteine motif (CCGPCC) was introduced into a surface loop region within the NTD of CA (amino acids 87 to 92) near the position previously used to label intact virus⁶⁰ to allow for FlAsH reagent binding. In Figure 2B, the position of a tetracysteine motif is indicated with the symbol for the FlAsH tag. The substrate containing MA and full-length CA domains with the tetracysteine motif in the NTD of the CA is termed MA/CA. The FlAsH reagent is a nonfluorescent biarsenical derivative of fluorescein that becomes fluorescent upon binding to its target,⁶¹ as illustrated in Figure 2A. In a protease assay employing FP, the enzyme activity is measured by a decrease in polarization values (mP) as the result of the change in size of the high-molecular weight fluorescent substrate to the low-molecular weight cleavage product (Figure 2A). Therefore, to increase differences in size before and after cleavage by the HIV-1 PR, the original MA/CA substrate was altered to include a fusion of a GST tag to the N terminus of MA and a truncation of the CTD of CA resulting in a protein substrate of 56 kDa (Figure 2A and B). This prototype substrate is designated as MA/CAΔ. In our FP assay, the size difference before and after proteolysis is approximately 41 kDa (56 kDa to 15 kDa; Figure 2A). The MA/CAΔ-Y132I substrate contains an additional Ile substitution at the P1 position of the MA/CA cleavage site to block cleavage at the MA/CA site by the HIV-1 PR.³⁷ These substrates include a His₆-tag at the N terminus of the protein to aid purification. When expressed in E. coli, the soluble form of the proteins were made in large quantity and the modifications and mutations made to the original MA/CA protein did not significantly affect the solubility of the proteins. We also created p15-C2S substrate to examine cleavage at an alternative Gag cleavage site, SP2/p6, in a gel-based secondary assay. This protein substrate contains the NC, SP2, and p6 domains with Cys to Ser substitutions in the zinc-finger motifs within the NC domain and a tetracysteine motif introduced within the C-terminal region of the p6 domain.

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Figure 2

Overview of the FP-based HIV-1 protease assay and schematic diagram of the MA/CA constructs used in the assay. (A) The FP assay detecting the changes in size of the fluorescently labeled protein upon proteolysis. The free FlAsH tag animated in gray increases in fluorescence after binding to a tetracysteine motif in the NTD of CA (CA_N), which is animated in black. The CA_N labeled with a FlAsH reagent is also shown in black. In the presence of the HIV-1 PR, the slowly rotating 56 kDa substrate releases the fast rotating 15 kDa CA_N resulting in a reduced FP value. (B) MA/CA-derived constructs in the E. coli expression vector pET30b. All of the constructs contain an intact MA domain and the CA_N. The MA/CA construct contains an intact MA domain and a full-length CA domain. A tetracysteine motif (CCGPCC) is inserted within the CA_N and the position of the tetracysteine motif is indicated with a FlAsH tag symbol. The gray boxes located either at the N-terminus or at the C-terminus of each construct represent a His6-tag. For MA/CAΔ constructs, a GST tag is fused to the N-terminus of the MA domain to increase the size of the protein. The p15 derived p15-C2S protein construct shows Cys→Ser mutations within the zinc-finger motifs in the NC domain and amino acid substitutions within p6 to create a tetracysteine motif. The cleavage sites between MA and CA, and between SP2 and p6 are indicated with a scissor graphic. The slow cleavage event at the NC/SP2 site is depicted with a small scissor.

FP-based HIV-1 Protease Assay Development in 96-well Plates

We have demonstrated previously that the efficiency of cleavage of the substrate MA/CAΔ (≅ 56 kDa) was not affected by the alterations made to the MA/CA protein (≅ 40 kDa) in a gel-based assay. As shown previously⁵⁷ and in Figure 3A, the two substrates, MA/CA and MA/CAΔ, are versatile tools that can be used to study HIV-1 proteolysis in a gel-based assay since the two full-length substrates and their cleavage products differ in size enabling the use of both substrates in the same proteolysis reaction. The full length CA, shown in open arrowhead in Figure 3A, is the cleavage product of the MA/CA substrate, and the truncated CA, CAΔ, shown in closed arrowhead in Figure 3A, is the cleavage product of the MA/CAΔ substrate. Given the equivalent cleavage rates of the modified and unmodified substrates, the more asymmetric substrate, MA/CAΔ, was used in a fluorescence polarization readout.

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Figure 3

Detection of the cleavage of the MA/CAΔ substrate labeled with a FlAsH tag by gel-based assay and FP assay in a 96-well plate. (A) Fluorescence image of the gel-based assay employing two substrates. MA/CA and MA/CAΔ were labeled with the FlAsH reagent in the same reaction, and proteolysis was performed by the addition of 50 nM HIV-1 PR at 30°C. Proteolysis was stopped at the various time points shown above the gel image for further SDS-PAGE analysis. The aliquot for 0 min time point was taken before the enzyme was added. Fluorescently labeled proteins were visualized by fluorescence imaging. Filled arrowheads indicate MA/CAΔ substrate and its cleavage product (CAΔ) and open arrowheads indicate MA/CA substrate and its cleavage product (CA). The domain shown in black (CA_N) indicates the location where the FlAsH reagent binding occurred. (B) Cleavage of the MA/CA site of the MA/CAΔ substrate measured by fluorescence polarization. End-point analysis shows reduced FP values in the presence of HIV-1 PR as a function of substrate concentration. Increasing amounts of MA/CAΔ (0 µM, 0.25 µM, 0.5 µM, 1 µM, 2 µM, and 4 µM) were incubated with 200 nM FlAsH reagent overnight, proteolysis was performed at 30°C for 3 h in the absence (open circle) or presence (closed circle) of 1 µM HIV-1 PR, and FP values were measured. (C) Time-point analysis of FP assay. The assay was performed in the presence of 2 µM substrate and 200 nM FlAsH reagent. Immediately after adding 50 nM PR, FP values were measured over 3 h-time period with 5-min intervals. The reading collected from the well in which protease was not added was considered as 0 min time point.

Fluorescence polarization is a convenient approach to measure the activity of a protease and is amenable to high-throughput screening in a plate format. To develop an assay to screen for small molecules that specifically block the MA/CA processing site, protease cleavage of the substrate MA/CAΔ was measured by changes in FP values in a 96-well plate. The optimum concentrations of FlAsH reagent and substrate for the FP assay were determined by titrating the FlAsH reagent with the substrate MA/CAΔ. The largest differences in FP values as a function of protease cleavage were observed with 200 nM FlAsH reagent (data not shown). The formation of a high-molecular-weight fluorescent complex of the FlAsH reagent bound to MA/CAΔ led to an increase in FP value until the FlAsH reagent was saturated with the substrate (Figure 3B). The FP value approached a plateau when substrate concentration was increased to 1 µM. Therefore, based on the titration data shown in Figure 3B, 2 µM substrate and 200 nM FlAsH were chosen as optimum concentrations for labeling the substrate for the FP assay. In the presence of 1 µM HIV-1 PR, the FP value was significantly reduced due to cleavage at the MA/CA site (Figure 3B). To define a protease concentration for time-point FP analyses, 2 µM substrate was incubated with different amounts of protease, and the result showed a useful concentration of protease to be 50 nM, allowing cleavage of the substrate over a convenient period of time, 3 h (data not shown). Under these conditions (substrate: 2 µM; FlAsH: 200 nM; HIV-1 PR: 50 nM), the time-point FP values of MA/CAΔ gradually decreased over time due to cleavage at the MA/CA site while the FP values in the absence of HIV-1 PR stayed high over a 3 h-time period, showing no changes compared to the initial value (Figure 3C).

Highly Sensitive FP-based HIV-1 Protease Assay

When a substrate containing the Y132I mutation to block MA/CA cleavage was used in a time-point FP assay, the FP values did not change over time (Figure 4A). After measuring the FP values, a portion of the FP assay reaction was analyzed by SDS-PAGE to confirm the state of the final cleavage products. The SDS-PAGE result shown by Coomassie staining revealed that the extent of cleavage of the substrates was consistent with the extent of proteolysis inferred from FP assay, and that the substrate and products were stable without displaying any detectable degradation (data not shown). The sensitivity of the assay was also tested in the presence of the HIV-1 PR inhibitor saquinavir (SQV). In the presence of increasing amounts of SQV from 1 nM to 25 nM, the changes in FP values decreased due to inhibition of PR activity by SQV (Figure 4B), although the 1 nM reaction includes a small mP effect caused by the presence of 4% DMSO (from the addition of SQV). In the presence of 25 nM SQV, the FP values stayed as high as those in the absence of HIV-1 PR suggesting that enzyme activity was essentially completely inhibited. Again, these FP data were confirmed by the SDS-PAGE analysis with Coomassie staining showing a gradual decrease in cleavage products, GST-MA and CAΔ, with increasing amounts of SQV (Figure 4C). Thus, these results demonstrate that the FP assay is sensitive for discerning changes in proteolytic processing as shown by the detectable change in FP in the presence of increasing amounts of a protease inhibitors.

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Figure 4

Highly sensitive FP-based HIV-1 protease assay. (A) FP values of the Y132I mutant substrate, MA/CAΔ-Y132I, in the presence of PR (Δ) and the MA/CAΔ substrate in the absence of PR (×) measured at 30°C for 3 h. (B) FP values of the MA/CAΔ in the presence of increasing amounts of the HIV-1 PR inhibitor, SQV, measured at 30°C for 3 h. After labeling reaction in the presence of 2 µM substrates and 200 nM FlAsH reagent, the indicated concentration of SQV and 50 nM protease were added, and FP values were measured. The FP values obtained from the reaction in the absence of both SQV and PR was added for comparison. (C) Coomassie Blue staining of the protein samples after FP measurements. The level of cleavage at each SQV concentration is shown. The uncleaved substrate, MA/CAΔ, is depicted as GST-MA-CA_N and two cleavage products are depicted as GST-MA and CA_N.

HTS Assay Validation in 384-well Plates

To screen a large number of compounds using an automated system in 384-well plates, we first optimized the FP assay for this format. HTS assay validation was then performed on the automated platform to determine the robustness and reproducibility of the assay by calculation of the Z'–values and CV. HTS assay validation included three independent runs on different days in which each run consisted of two 384-well plates, comprising a total of six plates. Each plate contained two columns of positive controls (red wells) and two columns of negative controls (green wells) having substrate plus wild-type PR and substrate plus heat-inactivated PR, respectively (Figure 5A). The wells shown in yellow contained the same reaction as in the positive control wells since no compound was tested in the HTS validation assay. The results of these validation experiments are summarized in the Table 1 showing an average CV less than 3% and 2 % for the positive controls and the negative controls, respectively, and an average Z'–value of 0.79. The data from the plate 1 is shown in Figure 5B, showing that FP values for the positive and the negative controls stayed constant across the plate with the average difference of approximately 140 mP between the two controls. For this particular plate, the CV was 2.29% for the positive controls and 1.25% for the negative controls, and the Z'–value was 0.83. These validation parameters indicate that the assay presented in this study is robust, reliable, and suitable for HTS to identify HIV-1 maturation inhibitors targeting the MA/CA cleavage site.

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Figure 5

HTS assay validation in 384-well plates. (A) Design of each 384-well plate tested for HTS validation of the assay. Individual 384-well plate includes two columns of positive controls shown in red and two columns of negative controls shown in green. The positive control wells contain MA/CAΔ substrate plus wild-type PR and the negative control wells contain MA/CAΔ substrate plus heat-inactivated PR. The reactions in the wells shown in yellow are the same as the one in the positive controls. The validations were performed on three different days, testing two 384-well plates on each day. (B) Well-to-well variation for the positive and negative controls shown in the scatter plot of the data from the plate 1. Data from the negative controls are denoted as −PR and data from the rest of the wells including the positive controls are denoted as +PR.

Assay Validation With the LOPAC¹²⁸⁰ Compound Library

To validate further the utility and reproducibility of the assay, we conducted a pilot screen using the LOPAC¹²⁸⁰ compound library (Sigma-Aldrich) comprising 1,280 small-molecule compounds with well-characterized pharmacological activities. Each compound was screened in duplicate at a final concentration of 10 µM. Each plate also contained two columns of positive controls (red wells) and two columns of negative controls (green wells) as shown in Figure 5A. No compound was added to the control wells, and heat-inactivated PR was used in the negative control wells. The values from these control wells were used to determine the percentage of inhibition for each compound tested, setting the mean values from the positive control wells as 0% inhibition and the mean values from the negative control wells as 100% inhibition. When the percentage of inhibition of all compounds tested in duplicate was plotted in Figure 6A, most of the compounds had no effect on cleavage of the substrate by HIV-1 PR with % inhibition values less than 20% of the mean value of the negative controls. In this screen, the two compounds, AC-93253 iodide and I-OMe tyrphostin AG538 (shown in circles; Figure 6A), had a readout of approximately 50% inhibition. However, results from the gel-based assay demonstrated that these two compounds had no inhibitory activity (data not shown). It is possible that these compounds caused protein aggregation resulting in relatively high FP values. Conversely, some compounds generated FP values lower than the mean value of the positive controls. We speculate that these small compounds are fluorescent themselves, contributing to the lower FP values of the reaction.

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Figure 6

Pilot screening using the LOPAC¹²⁰⁸ compound library. (A) The percent inhibition of the cleavage reaction of the MA/CAΔ substrate by the HIV-1 PR in the presence of each compound. Design of 384-well plates used for screening LOPAC¹²⁰⁸ compound library is the same as that used for HTS assay validation except that the wells shown in yellow contain a compound. Neither positive nor negative control wells contain a compound. Each compound was screened in duplicate and each dot represents the result from the individual compound. All the data from the duplicate screening denoted as testA and testB were plotted. Two compounds displaying approximately 50% of inhibitory activity are circled. (B) Bland-Altman plot of the difference between % inhibition of testA and % inhibition of testB plotted against the mean of % inhibition of testA and % inhibition of testB. The mean difference ± 2 standard deviations (95% limits of agreement) are shown with dashed lines.

To compare the two measurements from the LOPAC validation assay, the degree of agreement between the two measurements of the percent inhibition was assessed by using Bland-Altman analysis by plotting the differences between the two measurements against the averages of the two measurements (Figure 6B). The mean difference between two measurements was −0.36 and the 95% limits of agreement, defined as the mean difference ± 2 standard deviations, ranged from −11 to 12. This small range of limits suggests good agreement between two measurements. In addition, the plot shows no trend of systematic bias. Overall, this pilot screen using the LOPAC¹²⁸⁰ compound library confirmed the reproducibility, robustness, and suitability of this assay for HTS with an average Z'-value of 0.81 and average CV values of 0.02 and 0.01 for positive controls and negative controls, respectively.

We also tested the maturation inhibitor, bevirimat, in a 96-well plate for time-point analysis. The compound was tested at a final concentration of 10 µM, a concentration 1000-fold higher relative to the EC50 measured in cell culture. As expected given its specificity for inhibition of the CA/SP1 cleavage site, bevirimat did not show any significant inhibitory activity on cleavage at the MA/CA site in a gel-based assay (data not shown). In a FP assay, bevirimat had no inhibitory activity on cleavage at the MA/CA site beyond the small effect caused by the presence of 5% DMSO included in the reaction (data not shown).

We thank Matt Redinbo for assistance with the initial anisotropy measurements.

Funding Sources

This work was supported by NIH grants P01-GM066524, R21 NS073052, and R01 GM65347. This work was also supported by the UNC Center For AIDS Research (NIH award P30-AI50410) and the Lineberger Comprehensive Cancer Center (NIH award P30-CA16086).