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A PDZ domain-based assay for measuring HIV protease activity: Assay design considerations

Department of Automated Biotechnology, Merck Research Laboratories, North Wales, Pennsylvania 19454, USA

Reprint requests to: Marc Ferrer, Department of Automated Biotechnology, Merck Research Laboratories, 502 Louise Lane, North Wales, PA 19454, USA; e-mail: marc_ferreralegre{at}merck.com; fax (267) 305-3625.

(RECEIVED October 10, 2002; FINAL REVISION December 6, 2002; ACCEPTED December 9, 2002)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0235603.

	Abstract

TOP Abstract Introduction Results and Discussion Materials and methods References

 
We have recently described a biochemical detection method for peptide products of enzymatic reactions based on the formation of PDZ domain•peptide ligand complexes. The product sensor is based on using masked or cryptic PDZ domain peptide ligands as enzyme substrates. Upon enzymatic processing, a PDZ-binding motif is exposed, and the product sequence bound specifically by a Eu³⁺chelate-labeled GST–PDZ ([Eu³⁺]GST–PDZ). The practical applicability of this PDZ-based detection method is determined by the affinity of the PDZ domain•peptide ligand interaction, and the efficiency of the enzyme to process the masked peptide ligand. To expand the use of this PDZ-based detection strategy to a broader range of enzymatic assays, we have taken advantage of the plasticity in ligand recognition by the variety of PDZ domains found in nature. In the original work, the PDZ3 of PSD-95 was used, which preferentially recognizes the consensus sequence Ser-X-Val-COOH. Here, we show that NHERF PDZ1, which binds to the consensus sequence Thr/Ser-X-Leu-COOH, can be used to extend the flexibility in the recognition of the carboxy-terminal amino acid of the ligand, and monitor the enzymatic activity of HIV protease. The choices of detection format, for example, TRET or ALPHA, were also investigated and influenced assay design.

Keywords: PDZ domains; TRET; ALPHA; HIV protease

Abbreviations: ALPHA, Amplified Luminescence Proximity Homogeneous Assay • CFTR, cystic fibrosis transmembrane regulator • GST-PDZ, Glutathione-S-Transferase fused to PDZ domain • HIV-PR, human immunodeficiency virus protease • mAb, monoclonal antibody • PBS, phosphate buffered saline • PBST, PBS with tween-20 • PBSTB, PBS with tween-20 and BSA • NHERF, Na+/H+ exchanger regulatory factor • PDZ, PSD95/Discs-large/ZO-1 • PSD95, postsynaptic density 95 • SA, streptavidin • TRET, time resolved energy transfer • [XL665]SA, allophycocyanin-conjugated streptavidin • , hydrophobic residue • , aromatic residue

	Introduction

TOP Abstract Introduction Results and Discussion Materials and methods References

 
We have recently described a detection method utilizing the formation of PDZ domain•peptide ligand complexes as a new alternative to antibody-based detection systems (Ferrer et al. 2002). The assay was based on the concept of epitope unmasking or neoepitopes, which have been successfully used previously in conjunction with antibody-based assay detection strategies (Zuck et al. 1999; Li et al. 2000). Antibodies or other ligand-binding proteins, such as PDZ domains, capable of specific recognition of neoepitopes, are becoming critical reagents for the development of homogeneous in vitro assays.

PDZ are a family of small modular domains of 80–90 amino acids that bind to the carboxyl terminus of an interacting protein (Fanning and Anderson 1999; Hung and Sheng 2002). Recently, it has been estimated that 440 PDZ domains in 259 different proteins are present in the human genome (Hung and Sheng 2002). They comprise four classes, according to their specificity for the carboxy-terminal peptide ligand sequence. Class I recognizes the consensus sequence -X-Ser/Thr-X--OH; class II the consensus sequence -X--X--OH; class III recognizes the sequence -X-Glu/Asp-X--OH; and class IV the sequence -X-X--Glu/Asp-OH (Bezprozvanny and Maximov 2001; Vaccaro and Dente 2002). The affinity of these peptides for specific PDZ domains are reported to range from low nanomolar to mid micromolar (Songyang et al. 1997; Stricker et al. 1997; Hall et al. 1998; Niethammer et al. 1998; Short et al. 1998; Wang et al. 1998; Lin et al. 1999; Schneider et al. 1999; Beebe et al. 2000; Fuh et al. 2000; Gee et al. 2000; Wang et al. 2000; Harris et al. 2001; Raghuram et al. 2001). The different classes of PDZ domains indicate that, in general, the binding sites of PDZ domains are adaptable and have a certain degree of plasticity, while maintaining certain structural characteristics. However, each PDZ domain within a class shows a high degree of specificity for a particular residue in the consensus sequence. For example, PSD95 PDZ3 and NHERF PDZ1 both belong to class I. However, the PSD95 PDZ3 binds preferentially to the sequence -X-Ser-X-Val-OH, whereas the NHERF PDZ1 has been shown to bind with high affinity (18 nM) to the sequence -Asp-Thr-Arg-Leu-OH of the CFTR (Hall et al. 1998). The NHERF PDZ1 also interacts with other peptide motifs, including -Asp-Ser-Leu-Leu-OH of the ß2-adrenergic receptor and -Asp-Ser-Phe-Leu-OH of the platelet-derived growth factor receptor (PDGFR) (Hall et al. 1998; Maudsley et al. 2000; Karthikeyan et al. 2001a). These results suggest that the preferential consensus sequence recognized by the NHERF PDZ1 is -Asp-Ser/Thr-X-Leu-OH.

HIV-PR was chosen to further investigate the applicability of the PDZ domain-based strategy because this enzyme remains an important target for the development of anti-HIV therapeutics. Furthermore, HIV-PR has been characterized extensively, and numerous known inhibitors exist to validate the assay. Attempts to develop an HIV-PR assay based on the initial PDZ detection system with PSD95 PDZ3 were unsuccessful because either (1) potential HIV-PR peptide products did not have sufficient affinity for PSD95 PDZ3 or (2) potential HIV-PR substrates that would expose PDZ3 ligands were not processed by the enzyme. HIV protease requires a bulky hydrophobic amino acid (e.g., Leu, Phe, Tyr) in the substrate P1 position, that subsequent to hydrolysis, yields a peptide having low affinity for the PSD95 PDZ3, which optimally requires a Val at the carboxy-terminal position (Griffiths et al. 1992; Fig. 1). To further expand the applicability of the PDZ-based detection system, we explored the possibility of using NHERF PDZ1 as a product sensor of peptides terminating in -Leu-OH, therefore enabling detection of the HIV protease product.

View larger version (18K): [in this window] [in a new window]   Figure 1. HIV protease substrate and PDZ domain ligand overlap consensus sites. (A) General peptide backbone showing the nomenclature for PDZ ligands (Xn-1 sites) and protease substrates (P and P' sites) and the overlap of residues critical to HIV protease substrate processing (gray hatched spheres) and to productive PDZ ligand binding to PDZ domains (yellow solid spheres). (B) Alignment of PDZ ligand consensus sequences for a given PDZ domain, protease substrate cleavage sites, and derived assay peptide substrate. Initially, a HIV protease substrate was designed based on the PSD95 PDZ3 binding consensus sequence, but no product formation was detected, presumably because the optimal residue in the P1 position for HIV protease cleavage is a large hydrophobic or aromatic residue. Two alternative substrates were designed on the basis of work by Poorman et al. (1991), and the NHERF PDZ1 binding consensus sequence.

	Results and Discussion

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Detection of NHERF PDZ1 •peptide ligand interactions
Direct chemical labeling of the NHERF GST–PDZ1 with Eu³⁺-chelates resulted in a domain that did not bind to any of the peptide ligands (Table 1) possibly due to occlusion of the ligand-binding site or disruption of the tertiary structure of the domain. To avoid labeling the GST–PDZ, a Eu³⁺-chelate-labeled anti-GST mAb was used for detection. By use of this sandwich detection format, it was possible to detect binding of the CFTR tail peptide to the NHERF GST–PDZ1 with a signal-to-background ratio (at 5 nM biotin-peptide) of 10-fold. A similar signal-to-background ratio (15-fold) is obtained for the detection of the -ESSV-OH peptide by PSD95 GST–PDZ3, using the Eu³⁺-chelate-labeled anti-GST mAb format. However, no binding was detected between NHERF GST–PDZ1 and a peptide designed to be the product of an optimal HIV protease substrate, in which the -DTRL-OH sequence of CFTR is modified by a unique substitution, -DTVL-OH.

View larger version (22K): [in this window] [in a new window]   Figure 2. Fluorescence polarization data showing binding specificity for particular PDZ domains. Each peptide (10 nM) was probed by an increasing concentration of GST–PDZ. The peptides, Fluorescein-HRRSARY LESSV-OH (squares), Fluorescein-HRRNEEVQDTRL-OH (circles), and Fluorescein-HRRSARYLDTVL-OH (triangles) were probed by both the PSD95 PDZ3 (solid symbols) and the NHERF PDZ1 (open symbols). Error bars indicate standard error of the means for n = 3 or 6.

ALPHA technology provides an alternative detection mode to TRET. ALPHA relies on the use of donor and acceptor beads that are coated with a layer of hydrogel, providing functional groups for bioconjugation. When a biological interaction between molecules brings the donor and acceptor bead into proximity, a cascade of chemical reactions is initiated to produce an amplified signal. Upon laser excitation at 680 nm, a photosensitizer in the donor bead converts ambient oxygen to a reactive, excited singlet state (¹O₂). The ¹O₂ molecules diffuse to react with a chemiluminescer in the acceptor bead that further activates fluorophores contained within this bead. The fluorophores subsequently emit light at 520–620 nm. In the absence of a specific biological interaction, the singlet state oxygen molecules produced by the donor bead go undetected without the close proximity of the acceptor bead. Because the ligands are attached to a surface, there is potential for cooperative interactions between the binding partners, allowing the ALPHA detection method to potentially detect weaker interactions (micromolar range) compared with TRET. In addition, this technology can accommodate higher concentration of peptide ligands than TRET methods, further driving the binding equilibrium to the bound species. At a peptide concentration of 5 nM, ALPHA gave a very high signal-to-background ratio for the CFTR peptide binding to NHERF GST–PDZ1 (300-fold), and for the -ESSV-OH peptide to PDS95 GST–PDZ3 (750-fold) (Table 1).

Design of masked PDZ ligands as HIV-PR substrates
The design of a peptide substrate for the HIV-PR assay required a sequence that would be an optimal substrate for the enzyme, as well as an optimal PDZ ligand (Fig. 1) upon enzyme processing. Data from Poorman et al. (1991) provided a starting point in the substrate design, with an exhaustive statistical study on the probability of numerous substrates to be cleaved by HIV-PR. HIV-PR prefers the following 8-mer peptide sequence with cleavage occurring at the scissile bond between the 4^th and 5^th residue: (X)-(no )-(small)-( or )-cleavage-(/P)-(E/N)-(no )-(X), in which is a hydrophobic residue and is an aromatic residue (Fig. 1) (Poorman et al. 1991; Griffiths et al. 1992; Erickson and Eissenstat 1999). Furthermore, HIV-PR recognizes only - or -P at the scissile bond. Among the substrates tested that received a high rating for cleavage, was the sequence DTVL-EEMS-NH₂, with the cleavage occurring between the leucine and the glutamic acid. However, the product sequence DTVL-OH was not an optimal NHERF PDZ1 binder (Table 1). Therefore, the sequence DTRL-EEMS-NH₂ was first tested as a substrate for HIV-PR, because the product of the enzymatic cleavage was a stronger NHERF PDZ1 ligand (Table 1). However, the DTRL-EEMS-NH₂ sequence was not cleaved by HIV-PR, suggesting that the arginine residue could not be accommodated in the P₂ position (data not shown). The HIV-PR assay was therefore optimized using the initial substrate DTVL-EEMS-NH₂.

Optimization of the HIV-PR assay conditions
To accommodate the suboptimal NHERF PDZ1-binding partner unmasked by HIV-PR activity (-DTVL-OH), the TRET, and ALPHA detection conditions were optimized by titrating the amount of detection reagents, using 50 nM total biotinylated peptide, and mimicking 10% substrate turnover (data not shown). The optimal detection conditions for the TRET detection system were 10 nM NHERF PDZ1, 5 nM [Eu³⁺]anti-GST mAb and 12.5 nM [XL665]SA. In the ALPHA format, the maximum signal-to-background was detected when using 80 nM NHERF PDZ1, 20 µg/mL of both the acceptor anti-GST mAb beads and the donor SA beads.

To determine the lower detection limit and dynamic range of the ALPHA and the TRET assay, the biotin-product (biotin-HRRSARYLDTVL-OH) was titrated using the optimal detection conditions (determined above) for each system, while keeping the total amount of biotin-peptide, substrate plus product, constant at 50 nM. Representative saturation curves for the binding of the biotin-peptide to the NHERF GST–PDZ1 are shown for both ALPHA and TRET detection mode in Figure 3A. The lower detection limit for TRET is significantly lower (5 nM product or 10% turnover) than that of ALPHA (10 nM product or 20% turnover). Although the TRET detection mode appears to be more sensitive, providing higher signal-to-background ratios at conditions mimicking lower percent turnover by the enzyme, there is a critical point (30% turnover), in which the ALPHA detection mode provides significantly higher signal-to-background ratios (Fig. 3B). These results suggest that a threshold amount of biotin-product peptide must be generated for the ALPHA beads to optimize the avidity effects that amplify the signal caused by a protein–protein interaction. In our particular system, we do not appear to be saturating the beads at 50 nM biotin-product peptide (the dynamic range does not plateau significantly), however, with the TRET-based system, we do begin to deplete our detection reagents at 50 nM biotin-product peptide (the dynamic range does plateau). As illustrated in Figure 3, the dynamic range for ALPHA has a much steeper slope than the TRET mode, and the signal increases at a much faster rate than TRET after a certain threshold is reached. Due to the lower affinity of the PDZ ligand, the dynamic range is shifted to the right significantly, as more product peptide is required to reach a point at which the ligand binds the PDZ domain and signal is produced.

View larger version (19K): [in this window] [in a new window]   Figure 3. Detection limit and dynamic range for PDZ-based TRET and ALPHA assay. (A) The two curves correspond to the two detection systems tested, TRET (), and ALPHA (). Biotin-product was titrated at a constant total amount of biotin-peptide (50 nM) and TRET or ALPHA measured using the optimal conditions for each detection system. The saturation curves were fitted to one-site binding dose-response functions (GraphPad Prism Software). (B) The two curves correspond to the two detection systems tested, TRET (), and ALPHA (). Biotin-product was titrated at a constant total amount of biotin-peptide (50 nM) and TRET or ALPHA measured using the optimal conditions for each detection system. The Y-axis corresponds to the signal-to-background for each system, with the background being 50 nM biotin-substrate or zero percent turnover. The inlaid graph shows the data for <40% turnover on a smaller S/B scale. Final concentrations of reagents in the TRET detection mixture were as follows: 50 nM biotin-peptide, 10 nM GST–PDZ, 5 nM [Eu3+]anti-GST mAb, 12.5 nM and [XL665]SA. Final concentrations of reagents in the ALPHA detection mixture were as follows: 50 nM biotin-peptide, 80 nM GST–PDZ, and 20 µg/mL of each anti-GST mAb-acceptor and SA-donor beads.

View larger version (23K): [in this window] [in a new window]   Figure 4. (A) Time course of Biotin-HRRSARYLDTVLEEMS-OH (1 µM) hydrolysis by HIV-PR at increasing concentrations of enzyme () 10 nM; () 5 nM; () 2.5 nM; () 1 nM; (•) 0.5 nM; () no enzyme as detected by TRET technology (error bars indicate standard error of the means, n = 3). (B) Inhibition of HIV-PR (1 nM) by several known inhibitors; () Indinavir; () Compound A; (•) Compound B. (error bar indicates range for n = 2). Final concentrations of reagents in the detection mixture were as follows: 50 nM biotin-peptide, 10 nM GST–PDZ, 5 nM [Eu3+]anti-GST mAb, 12.5 nM, and [XL665]SA.

View larger version (19K): [in this window] [in a new window]   Figure 5. (A) Time course of Biotin-HRRSARYLDTVLEEMS-OH (1 µM) hydrolysis by HIV-PR at increasing concentrations of enzyme () 10 nM; () 5 nM; () 2.5 nM; () 1 nM; (•) 0.5 nM; () no enzyme as detected by ALPHA technology (error bars indicate standard error of the means, n =3). (B) Inhibition of HIV-PR (2.5 nM) by several known inhibitors; () Indinavir; () Compound A; (•) Compound B. The inhibition curves could be fitted to a one-site binding function. (error bar indicates range n = 2). Final concentrations of reagents in the detection mixture were as follows: 50 nM biotin-peptide, 80 nM GST–PDZ, and 20 µg/mL of each anti-GST mAb acceptor and SA-donor beads.

Although affinity selection of peptide libraries remains a tool for the future development of this detection scheme (Songyang 1997; Stricker 1997; Wang et al. 1998; Fuh et al. 2000; Rosse et al. 2000), new innovations appear to be expanding the possibilities for the application of this novel detection strategy. To this end, altering the binding preference of PDZ domains to recognize new target sequences has been demonstrated through point mutations (Schneider et al. 1999; Gee et al. 2000; Vaccaro et al. 2001; Reina et al. 2002), allowing the possibility to use an optimal enzyme substrate without compromising the binding affinity of the product.

Here, we evaluated two detection technologies: TRET and ALPHA. In a comparison using a mixture of substrate and product from enzymatic reactions, TRET provided a lower limit of detection but narrower dynamic range than ALPHA. At 30% substrate turnover, both formats gave approximately the same signal-to-background ratio, therefore leading to the use of these conditions for the enzymatic reaction. In summary, for this PDZ-based detection system, both technologies provided similar results. However, for the purpose of high-throughput screening, TRET detection was more reproducible, required less GST–PDZ1 protein to detect binding, and required shorter incubation times.

By tapping the natural diversity of PDZ domains, we have expanded this biological detection strategy beyond our initial proof-of-principle study. We envision that highly unique recognition domains based on the PDZ scaffold architecture, but not found in nature, will be possible with site-mutagenesis or protein evolution and panning methods.

	Materials and methods

TOP Abstract Introduction Results and Discussion Materials and methods References

 
General
Biotin and Fluorescein-labeled peptides were purchased from Research Genetics, Princeton Biomolecules, or Synpep. Peptides with Tyr in the sequence were dissolved in DMSO to 1 mM solutions (concentration was determined by UV absorbance at 280 nm, using a _Tyr=1280 mole^-1Lcm^-1). Fluorescein peptides were dissolved in DMSO to 1 mM solutions (concentration was determined by UV absorbance at 492 nm, using a _FL = 66,000 mole^-1Lcm^-1). Otherwise, the concentration was estimated from dry weight. Molecular Probes HIV-PR substrate 1 was purchased from Molecular Probes. Streptavidin-XL665 (molar ratio: 1.9 XL665/ Streptavidin); SA-donor beads and anti-GST mAb-acceptor beads for ALPHA technology were from Perkin-Elmer. [Eu⁺]Anti-GST chelate mAb was obtained from PerkinElmer. HIV protease (HIV-PR) was provided by Dr. Lawrence Kuo (Merck Research Laboratories, West Point, PA), and HIV-PR inhibitors were provided by the Medicinal Chemistry Department (Merck Research Laboratories, West Point, PA).

TRET detection was carried out in 384-well, small volume, black, low-binding, microplates plates (Greiner). Assay plates were read using a Victor²V microplate analyzer (Perkin Elmer), with 340-nm excitation. Fluorescence emission was measured at 665 nm for FRET signal and 615 nm for Eu³⁺-chelate, and results are expressed as the ratio of fluorescence intensities, FI_665nm/FI_615nm. ALPHA detection was carried out in 384-well, small volume, white Proxiplates (Perkin-Elmer). Assays plates were read using an AlphaQuest microplate reader (Perkin-Elmer).

Subcloning and expression of GST–PDZ domains
The GST–PDZ domain corresponding to the PDZ module 3 of PSD-95 was prepared as described previously (Ferrer et al. 2002). The GST–PDZ domain fusion protein corresponding to the PDZ module 1 of rabbit NHERF was obtained by standard cloning and expression methods. Briefly, the NHERF PDZ1 (Karthikeyan et al. 2001a) coding sequence (Leu 11 to Leu 99, inclusive) was amplified from a plasmid of full-length rabbit NHERF (gift of Professor Richard Premont, Duke University, NC) using PCR with the primers 5'-CGGGATCCCTGCCCCGGCTCTGCTGC and 5'-GGAAT TCCAGCTGCTCGTCCGTCTCGGGGTC. The DNA was subcloned into the bacterial expression vector pGEX-2TK (Pharmacia) using the BamH1 and EcoR1 restrictions sites. The GST–PDZ was expressed in Escherichia coli BL21(DE3) using standard bacterial expression methods (Smith and Johnson 1988), and purified using reduced glutathione agarose beads (Molecular Probes) following protocols provided by the supplier.

Labeling of PDZ domains with Eu³⁺-chelate
PDZ and GST–PDZ domains were labeled with Eu³⁺-chelate isothiocyanate (Eu³⁺ W1024 ITC Chelate) from Perkin Elmer, as described before. Briefly, the protein solutions were concentrated to 3 mg/mL in PBS, and 50 µL (160 µg, 4.3 nmole) of concentrated solution added to lyophilized Eu³⁺-chelate isothiocyanate (100 µg, 140 nmole), and the reaction allowed to proceed for 16 h at 4°C. The reaction solution was applied to a Nick column (Amersham Pharmacia), and the fraction with labeled protein (400 µL in PBS) applied to a PD-10 column (Amersham Pharmacia). Labeled protein was eluted in 400-µL fractions with PBS. Fractions with protein were pooled. Protein concentration was quantified by Bradford Protein assay, and protein-bound Eu³⁺-chelate was quantified with DELFIA enhancement solution for Eu³⁺, and Eu³⁺ standard (Perkin Elmer).

Detection of PDZ•ligand interaction by TRET or ALPHA
Peptide ligands for the NHERF PDZ1 were initially tested for binding to the GST–PDZ directly labeled with Eu³⁺-chelate. To determine the optimal detection conditions giving maximum signal-to-background ratio, the amounts of [Eu³⁺]GST–PDZ (20 µL in PBSTB) and [XL665]SA (20 µL in PBSTB) were titrated simultaneously, at a constant concentration of biotin-peptide (5 nM final concentration, 10 µL in PBSTB). Direct labeling of the NHERF GST–PDZ1 produced a labeled protein that did not bind to any of the potential peptide ligands tested.

The interaction of the peptide ligands with the NHERF PDZ1 was also tested by indirectly labeling GST–PDZ with a [Eu³⁺]anti-GST mAb (Perkin Elmer). The Eu³⁺-labeled anti-GST allowed us to label the GST–PDZ1 without structurally modifying the PDZ1 domain itself (avoiding direct coupling to free Lys). A 10-nM biotin-peptide and 10-nM GST–PDZ domain were incubated together for 1 h (20 µL in PBSTB), followed by addition of a detection mixture (20 µL in PBSTB), comprised of the labeled components of the assay (10 nM [Eu³⁺]anti-GST mAb, and due to the tetrameric nature of streptavidin (SA), 2.5 nM [XL665]SA). The final mix was incubated at room temperature and read on the Victor²V.

To determine the dynamic range of the detection and the lower detection limit for the TRET assay, the amount of biotin-product peptide was increased while keeping the total amount of peptide constant at 50 nM. After incubating 5 µL of the biotin-peptide mix (200 nM) with 5 µL of the GST–PDZ (40 nM), 10 µL of [Eu³⁺] anti-GST (10 nM) and [XL665]SA (25 nM) detection mix is added and allowed to incubate at room temperature for 1 h, and then read on the Victor²V.

ALPHA technology was also investigated as a means to detect binding of potential peptide ligands to NHERF GST–PDZ1. To probe the interaction of the biotinylated peptides and the GST–PDZ1, SA donor beads were used to capture the biotinylated peptide, and anti-GST-labeled acceptor beads were used to capture the GST–PDZ1. A 10-nM biotin-peptide and 10-nM GST–PDZ1 were incubated for 1 h (10 µL in PBSTB), followed by addition of the detection mixture (10 µL in PBSTB), comprised of the beads mixture (40 µg/mL streptavidin donor and 40 µg/mL anti-GST acceptor beads). The plates were sealed and incubated in the dark at 25°C for 3 h before being read on the ALPHAquest reader.

To determine the dynamic range of the detection and the lower detection limit for the ALPHA assay, the amount of biotin-product peptide was increased while keeping the total amount of peptide constant at 50 nM. After incubating 5 µL of the biotin-peptide mix (200 nM) with 5 µL of the GST–PDZ (320 nM), 10 µL of the ALPHA bead mix (40 µg/mL each bead type) was added and allowed to incubate in dark at room temperature for 3 h, and then read on the ALPHAquest.

HIV protease assay using TRET technology
For the initial time courses, 50 µL of Biotin-HRRSARYLDTVLEEMS-NH₂ (2 µM in 0.1 M NaOAc, 1 M NaCl, 1 mM EDTA, 1 mM DTT, 1 mg/mL BSA at pH 4.7) was mixed with 50 µL of HIV-PR (2x in assay buffer), and the reaction let to proceed at 37°C. The enzymatic reaction was quenched by dilution of 5 µL of the enzymatic solution into 20 µL of PBSTB. For the detection step, 5 µL of quenched enzymatic reaction solution was added to 5 µL of NHERF GST–PDZ1 (40 nM in PBSTB) and incubated at 25°C for 10 min. Then, 10 µL of TRET detect mix (10 nM anti-GST Eu³⁺ and 25 nM [XL665]SA) were added. The reaction was incubated for 1 h at 25°C, and then read on the Victor²V.

For protease inhibition experiments, 0.25 µL of inhibitor was added to 20 µL of HIV-PR (2 nM in assay buffer), and after incubating for 10 min, the reaction was initiated by addition of 20 µL of peptide substrate (2 µM in assay buffer). The reaction was quenched after 30 min at 37°C by diluting 1/5 in PBSTB (5-µL reaction solution in 20 µL of PBSTB). For the detection step, 5 µL of diluted enzymatic reaction solution was added to 5 µL of NHERF GST–PDZ1 (40 nM in PBSTB) and incubated at 25°C for 10 min. Then, 10 µL of TRET detection (10 nM anti-GST Eu³⁺ and 25 nM [XL665]SA) were added. The reaction was incubated for 1 h at 25°C, and then read on the Victor²V. Background was measured from a reaction mixture with substrate, detection mixture, and no enzyme. IC₅₀ were estimated by fitting the data by nonlinear regression of a Klotz plot [ratio vs. log (Inhibitor)] using the Prizm curve-fitting software (GraphPad Software).

HIV protease assay using ALPHA technology
For the initial time courses, 50 µL of biotin-HRRSARYLDTVL EEMS-NH₂ (2 µM in 0.1 M NaOAc, 1 M NaCl, 1 mM EDTA, 1 mM DTT, 1 mg/mL BSA at pH 4.7) was mixed with 50 µL of HIV-PR (2x in assay buffer), and the reaction let to proceed at 37°C. The enzymatic reaction was quenched by dilution of 5 µL of the enzymatic solution into 20 µL of PBSTB. For the detection step, 5 µL of diluted enzymatic reaction solution was added to 5 µL of NHERF GST–PDZ1 (320 nM in PBSTB) and incubated at 25°C for 10 min. Then, 10 µL of ALPHA bead mix (40 mg/mL SA-donor beads and anti-GST mAb acceptor beads) were added. The reaction was incubated for 3 h at 25°C, in the dark, then the plate was dark and temperature adapted inside the AlphaQuest instrument for 10 min, and finally the signal was read.

For protease inhibition experiments, 0.25 µL of inhibitor was added to 20 µL of HIV-PR (5 nM in assay buffer), and after incubating for 10 min, the reaction was initiated by addition of 20 µL of peptide substrate (2 µM in assay buffer). The reaction was quenched after 30 min at 37°C by diluting 1/5 in PBSTB (5-µL reaction solution in 20 µL of PBSTB). For the detection step, 5 µL of diluted enzymatic reaction solution was added to 5 µL of NHERF GST–PDZ1 (320 nM in PBSTB) and incubated at 25°C for 10 min. Then, 10 µL of ALPHA bead mix (40 mg/mL SA-donor beads and anti-GST mAb acceptor beads) were added. The reaction was incubated for 3 h at 25°C, in the dark, then the plate was dark and temperature adapted inside the ALPHAQuest instrument for 10 min, and finally the signal was read. Background was measured from a reaction mixture with substrate, detection mixture, and no enzyme. IC₅₀ were estimated by fitting the data by nonlinear regression of a Klotz plot [counts vs. log (Inhibitor)] using the Prizm curve-fitting software (GraphPad Software).

HIV protease assay using a QFRET assay
For protease inhibition experiments, 0.25 µL of inhibitor was added to 20 µL of HIV-PR (4 nM in assay buffer), and after incubating for 10 min, the reaction was initiated by addition of 20 µL of peptide substrate R-E(EDANS)-S-Q-N-Y-P-I-V-Q-K (DABCYL)-R (HIV-PR Molecular Probes Substrate 1, Molecular Probes) (20 µM in assay buffer). The reaction was quenched after 30 min at 37°C by adding 20 µL of 1 M Tris. The plate was then read on the Victor²V with excitation set to 340 nm and emission set to 510 nm. Background was measured from a reaction mixture with substrate and no enzyme.

Determination of binding affinities (K_d) using fluorescence polarization
A total of 1 µL of fluorescein-labeled peptide (200 nM in PBSTB) was added to 19 µL of PDZ domain solution in PBSTB, in a 384-well black Optiplate from Packard. The reaction was incubated for 2 h at 25°C, and fluorescence polarization read using a Victor²V microplate analyzer. Binding affinities (K_d) were estimated by fitting the data by nonlinear regression of a Klotz plot [mP vs. log (GST–PDZ)] using the Prizm curve-fitting software (GraphPad Software).

	Acknowledgments

 
We thank Dr. Lawrence Kuo for supplying us with HIV-PR, the Medicinal Chemistry Department at MRL West Point for providing HIV-PR inhibitors, Chris Culberson for help with modeling the NHERF PDZ1-peptide ligand interactions, Professor Richard Premont for supplying us with rabbit NHERF construct, Dr. David Olsen for advice, and Dr. B. Strulovici for support and resources.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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