Luminometric method for screening retroviral protease inhibitors

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 345 (2005) 96–101 www.elsevier.com/locate/yabio

Luminometric method for screening retroviral protease inhibitors Dana Horáková a, Michaela Rumlová a,b, Iva Pichová b,¤, Tomán Ruml a,b,¤ a

b

Department of Biochemistry and Microbiology, Institute of Chemical Technology, Technická 3, 166 28 Prague, Czech Republic Department of Biochemistry, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 166 10 Prague, Czech Republic Received 14 April 2005 Available online 28 July 2005

Abstract We have developed a sensitive luminometric assay for determining the activity of retroviral proteases that uses proteolytic cleavage of polypeptide substrate immobilized on Ni–NTA HisSorb Strips microplates. The protease substrate derived from the Gag precursor protein of Mason–PWzer monkey virus (M-PMV) was conjugated with horseradish peroxidase (HRP), which catalyzes oxidation of luminol in the assay. The cleavage of the substrate was monitored as a decrease in luminescent signal caused by the release of the cleavage product conjugated to HRP. Testing of a set of M-PMV protease inhibitors conWrmed that this method is suYciently sensitive and speciWc for high-throughput screening of retroviral protease inhibitors.  2005 Elsevier Inc. All rights reserved. Keywords: Retroviral protease; Inhibitors; Luminescent assay; High-throughput screening

Retroviral proteases (PRs)1 mediate the cleavage of polyprotein precursors into structural proteins and enzymes. This process launches structural changes that result in particle reorganization and formation of a retroviral core and is a prerequisite for the virus infectivity. Several inhibitors that eYciently target the active site of HIV-1 PR have reached clinical application [1–6]. However, systematic screening and de novo design of inhibitors of the active site of PR and inhibitors targeting

* Corresponding authors. Faxes: +420 220183556 (I. Pichová), +420 220445140 (T. Ruml). E-mail addresses: [email protected] (I. Pichová), tomas. [email protected] (T. Ruml). 1 Abbreviations used: PR, retroviral protease; HRP, horseradish peroxidase; M-PMV, Mason–PWzer monkey virus; MA, matrix; PP, phosphoprotein; PCR, polymerase chain reaction; LB, Luria–Bertani; IPTG, isopropyl-
-D-thiogalactopyranoside; EDTA, ethylenediaminetetraacetic acid; RP-HPLC, reversed-phase high-pressure liquid chromatography; BSA, bovine serum albumin; RLU, relative luminescent units; CA, capsid; NC, nucleocapsid; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; MAV, myeloblastosis-associated virus; Sap, secreted aspartic proteases.

0003-2697/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.07.013

other potential sites important for PR activity as dimerization interface are needed to overcome the high mutation rate and subsequent emergence of drug-resistant strains of HIV-1. Several assays for the determination of the activity of PRs and testing of inhibitors have been established. The majority of these assays are based on trans- or autocatalytic cleavage of reporter proteins in bacteria or yeasts or on in vitro hydrolysis of substrates. Macromolecules such as viral polyproteins Gag/Gag-Pol, engineered heterologous proteins, and peptides mapping the cleavage sites in viral polyproteins are commonly used as substrates. The product formations are subsequently detected by chromatographic, electrophoretic, radiometric, spectrophotometric, Xuorometric, and colorimetric methods (for a review, see [7]). These assays are less suitable for high-throughput screening of inhibitors and are usually used for detailed enzymological studies. The introduction of acceptor and donor molecules at the ends of a peptide substrate enabled establishment of Xuorogenic assays based on the intramolecular Xuorescence resonance energy transfer and improved their use

Luminometric method for retroviral protease inhibitors / D. Horáková et al. / Anal. Biochem. 345 (2005) 96–101

for the high-throughput testing of PR activities [8–10]. Colorimetric assays were also developed for the screening of PR activity. Although the assay using isatin, which requires boiling of samples, is not suitable for protocols that use microtiter plates [11], the assay, which uses two nonenzymatic steps following enzymatic hydrolysis of a synthetic peptide, was shown to be applicable for high-throughput screening of HIV-1 PR activities [12]. However, this assay is laborious, and some components during the procedure are light and temperature sensitive. Assays based on the trans-cleavage of protease hydrolyzable sites introduced into the selective biological markers, such as a galactokinase [13],
-galactosidase [14], a tetracycline resistance protein [15], and a -repressor [16–18], were tested for screening of protease inhibitors. The activity of inhibitors is screened here as a loss of function of these biological markers. The method presented in this article is based on the luminometric detection of the activity of horseradish peroxidase (HRP) conjugated to the sensitive Mason– PWzer monkey virus (M-PMV) protease substrate, which is represented here by the polypeptide consisting of the matrix (MA) domain and 19 amino acids from the adjacent phosphoprotein (PP) domain of M-PMV Gag polyprotein. The cleavage of this substrate with M-PMV protease is monitored as a decrease of luminescent signal.

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with 100 g/ml ampicillin and grown overnight at 37 °C. The culture was then diluted 1:400 with the same medium and incubated to an optical density of OD600 »0.5, and protein expression was induced by the addition of isopropyl-
-D-thiogalactopyranoside (IPTG) to a Wnal concentration of 0.1 mM. The cells were harvested after 4 h by centrifugation at 5000g for 20 min in a Beckman JA-14 rotor. The cells were solubilized in buVer A (50 mM Tris (pH 8.0), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 2 mM ME, and 100 mM NaCl), lysed by lysozyme (0.2 mg/ml) for 30 min, and treated with sodium deoxycholate to a Wnal concentration of 0.1%. DNAse (1 g/ml) and RNAse (2 g/m) were added to the solution, and the solution was incubated for 30 min at room temperature. The solution was sonicated 4 £ 25 s on ice and centrifuged at 10,000g for 20 min in a Beckman JA-18 rotor. The supernatant containing the MA-PP19-His6 protein was dialyzed against buVer B (50 mM phosphate buVer (pH 7.0), 300 mM NaCl, and 10 mM imidazole). The Ni–NTA–agarose equilibrated with buVer B was added batchwise to the protein sample and shaken for 1 h at 4 °C. The agarose with immobilized MA-PP19-His6 was transferred to a column and washed with 50 mM phosphate buVer (pH 6.2) containing increasing concentrations of imidazole (10–100 mM). The MA-PP19-His6 protein was eluted by 50 mM phosphate buVer (pH 6.2) containing 250 mM imidazole. Synthesis of inhibitors

Materials and methods Cloning and expression of a protein substrate All DNA manipulations were carried out using common cloning techniques as described in [19], and the plasmid was propagated in Escherichia coli DH5. The PCR fragment encoding the M-PMV MA domain with 19 adjacent amino acids from the phosphoprotein (PP19) at the C-terminus was ampliWed using the following primers: 5⬘MA, CTTGCGCTCGCATATGG GGCAAG (carrying NdeI site); 3⬘PP, GAGAGGTC TTCTCGAGGTCTGTTTG (carrying XhoI site); and GAG-pGEMEX as a template. The resulting polymerase chain reaction (PCR) product was digested with NdeI and XhoI and ligated in frame with the sequence encoding the 6£ His tag into pET22b (Novagen). The resulting recombinant plasmid was named pEMAPP19His. The clone was characterized by restriction analysis and veriWed by DNA sequencing. Expression and puriWcation of MA-PP19-His6 Escherichia coli BL21(DE3) cells were transformed with pEMAPP19His. A single colony was used to inoculate 10 ml Luria–Bertani (LB) medium supplemented

The statine inhibitors were synthesized by an established solution method as described previously [20–22]. Crude inhibitors were puriWed by reversed-phase highpressure liquid chromatography (RP-HPLC), and their homogeneities were conWrmed by isocratic RP-HPLC, amino acid analysis, and high-resolution fast atom bombardment mass spectrometry. Conjugation of the substrate to HRP A two-step glutaraldehyde conjugation of HRP to MA-PP19-His6 protein was employed. HRP (10 mg) was incubated in 200 l of 1.25% glutaraldehyde in 100 mM sodium phosphate buVer (pH 6.8) for 18 h at room temperature. The unbound glutaraldehyde was removed from the solution by gel Wltration on Sephadex G-100. The brownish fractions, containing activated HRP, were collected and dialyzed against buVer C (100 mM sodium carbonate–sodium bicarbonate buVer, pH 9.5) containing 30% sucrose. The HRP solution was concentrated to 10 mg/ml and dialyzed against buVer C. Then 1 ml of activated HRP (10 mg/ml) was mixed with 100 l of MAPP19-His6 (5 mg/ml), and the pH of the solution was adjusted to 9.1. The conjugation proceeded in the presence of 0.15 M NaCl for 24 h at 4 °C. The Wnal product,

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Luminometric method for retroviral protease inhibitors / D. Horáková et al. / Anal. Biochem. 345 (2005) 96–101

HRP-MA-PP19-His6, was treated for 2 h with 100 l of 0.02 M ethanolamine at pH 7.0 and 4 °C, and the concentration of the conjugate was determined by the methods of both Bradford [23] and Lowry et al. [24]. Immobilization of the substrate HRP-MA-PP19-His6 was immobilized on Ni–NTA HisSorb Strips (Quiagen) for 4 h at room temperature in binding buVer (50 mM phosphate buVer (pH 7.3) containing 150 mM NaCl). To prevent adsorption of the protein to the wall, a 0.2% solution of bovine serum albumin (BSA) was added to each well. The declared binding capacity of Ni–NTA HisSorb Strips is approximately 20 pmol/well for small peptides (20–30 mers) and approximately 2–10 pmol/well for proteins, according to the manufacturer’s manual. Therefore, 550 ng of HRP-MA-PP19-His6 (MW » 55 kDa) in 100 l of binding buVer per well was used initially, and the plate was gently shaken overnight at 4 °C. Each well was washed four times with a washing buVer (50 mM phosphate buVer (pH 7.0) containing 300 mM NaCl). The washing buVer was removed for luminometric detection, and the wells were dried by tapping on paper towels. Luminescence emission study Next, 100 l of Britton–Robins buVer (pH 9.15, prepared from 40 mM phosphoric acid, 40 mM phenylacetic acid, 40 mM boric acid, and 200 mM sodium hydroxide) per well was pipetted with immobilized HRP-MA-PP19His6, and the plate was incubated at 37 °C for 30 min before adding 50 l of the stock solution of luminol-containing hydrogen peroxide (1 mg luminol in 12 ml H2O and 72 l H2O2) to each well. The intensity of emission light was recorded by Labsystems Luminoscan RT at 425 nm for 60 s as relative luminescent units (RLU). The data were evaluated with the commercial software package provided with the apparatus. Determination of the proteolytic activity of PR The M-PMV protease was prepared as described previously [25]. First, 100 l of a 4.6-M solution of the 13-kDa form of M-PMV PR in PR buVer (0.1 M phosphate buVer (pH 6.2) containing 150 mM NaCl) was added to each well with immobilized substrate (HRP-MA-PP19-His6) and incubated for 2 h at 37 °C. The cleavage products and protease were removed, and the wells were washed with the universal buVer (pH 9.15, prepared from 0.04 M phosphoric acid, 0.04 M acetic acid, and 0.04 M boric acid). The residual luminescence was detected after the addition of 100 l universal buVer and 50 l luminol solution (as described above).

Testing the inhibitors of M-PMV PR To screen and compare activities of diVerent inhibitors, 95 l PR buVer and 2 l inhibitor (100 nM) per well were pipetted with immobilized HRP-MA-PP19-His6. Then 3 l M-PMV PR (4.6 M) was added to each well, and the solutions were incubated at 37 °C for 30 min. The wells were washed with the universal buVer (pH 9.15), and the residual luminescence was measured in the presence of luminol solution as described above.

Results and discussion Substrate design PRs cleave Gag-related precursor proteins into structural proteins and enzymes. Sequences between individual domains in the Gag polyproteins represent natural cleavage sites, and peptides derived from these sequences are commonly used for detection of activities and substrate speciWcities of PRs. In M-PMV, the Gag polyprotein contains, in addition to the conservative MA, capsid (CA), and nucleocapsid (NC) domains, a PP domain and a p12 domain, both of which are located between MA and CA [26]. The peptide of the sequence QVM*AAV, which was derived from the MA-PP cleavage site, was shown to be a good substrate for M-PMV PR [27]. MA and PP were also eYciently released during the proteolytic processing of M-PMV immature capsids in vitro [28]. Therefore, this fragment, which is composed of the MA and an adjacent sequence from PP, was selected as a substrate for M-PMV PR in a luminometric assay. Luminescent analysis of the conjugated substrate The plasmid-containing sequences encoding the MA protein in fusion with the 19 N-terminal amino acids of PP and a C-terminal histidine tag (MA-PP19-His) was prepared and used for expression in E. coli under control of the phage T7 promoter. The MA-PP19-His6 protein was expressed in E. coli in soluble form and represented approximately 40% of the total cellular proteins. The fusion protein was puriWed to homogeneity using an Ni–NTA–agarose column and was conjugated to HRP in the presence of glutaraldehyde through lysine residues present within MA-PP19. The average concentration of the conjugate (HRP-MA-PP19-His6), detected by the methods of both Bradford [23] and Lowry et al. [24], was 40 g/ml. The concentration of HRP-MA-PP19-His6 and the time required for immobilization on microplates were optimized by the detection of HRP activity in the presence of hydrogen peroxide and luminol. The oxidation of luminol in basic conditions, catalyzed by HRP, yielded 3-aminophthalate, that is, an excited light-emit-

Luminometric method for retroviral protease inhibitors / D. Horáková et al. / Anal. Biochem. 345 (2005) 96–101

Fig. 1. Time course of proteolytic cleavage of immobilized HRP-MAPP19-His6 substrate by M-PMV protease. First, 100 l of 4.6 M MPMV PR in 0.1 M phosphate buVer (pH 6.2, containing 150 mM NaCl) was incubated for the desired time with the immobilized protease substrate on the HisSorb Strips plates at 37 °C. The cleavage product was washed out, and luminescence was recorded in the presence of luminol at 425 nm. Average values calculated from six series of measurements, including mean error, are included. RLU for universal buVer (pH 9.15) is 16 § 3.5.

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Fig. 2. Saturation curve for binding of HRP-MA-PP19-His6 on HisSorb Strips plates. Immobilization of increasing concentration of the conjugate HRP-MA-PP-His6 on the plate in the presence of luminol and hydrogen peroxide was monitored by measurement of the emitted light at 425 nm as RLU.

The time course of the cleavage of MA-PP19-His6 on Ni–NTA HisSorb Strips with M-PMV protease was monitored over 100 min, and the results show that the substrate was completely cleaved within 60 min (Fig. 2). Testing of inhibitors of PR

ting product. The luminescent signal reached its maximum at a conjugate concentration of 800 ng/ml (Fig. 1), and this concentration was selected as a standard concentration for immobilization of the protease substrate in the luminometric assay. The deviation of the amount of immobilized protein, tested in Wve independent experiments, was §10% from the average value. Protease activity determination The cleavage of the substrate MA-PP19-His6 with M-PMV protease was tested at pH 6.2 in the PR buVer (see Materials and methods), and the products were detected by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The cleavage of the fusion protein into the MA protein and the N-terminal domain of PP was veriWed by protein sequencing on a Procise amino sequencer (data not shown). The activity of MPMV PR was tested using the immobilized HRP-MAPP19-His6 on Ni–NTA HisSorb Strips plates at pH 6.2 in the presence of higher ionic strength (0.5 M NaCl), which promotes the proteolytic cleavage [27]. The average light emission of immobilized conjugate was detected as 380 § 21.4 RLU. Theoretically, up to 10 molecules of HRP can be conjugated through Lys residues to the MA domain and only 1 molecule of HRP can be conjugated to the N-terminal part of the PP. Thus, the release of the MA domain from MA-PP19 after cleavage with the PR, followed by its removal from the plates by washing plates with the universal buVer (pH 9.15), signiWcantly reduces the number of immobilized HRP molecules and consequently causes the decrease in the luminescent signal.

To test the applicability and selectivity of this method for inhibitor screening, several compounds available in our laboratory were used for inhibition of M-PMV PR. These inhibitors were originally designed for proteases of HIV, myeloblastosis-associated virus (MAV), and secreted aspartic proteases (Sap) of Candida spp. [20–22]. The inhibitors of deWned concentration were pipetted to the immobilized substrate, and its cleavage was evaluated 30 min after the addition of the protease solution. The results, summarized in Table 1, demonstrate that clinical inhibitors of HIV-1 PR Indinavir (In 1), NelWnavir (In 2), Ritonavir (In 4), Saquinavir (In 6), the inhibitor No. 5 (also designed for HIV-1 PR), and pepstatin A (In 7, a representative of speciWc inhibitors of aspartic proteases) inhibited M-PMV PR by approximately 35% (calculated from the decrease of RLU). Sap’s inhibitors (In 8–12) containing statine and phenylstatine isosteric groups originally designed for aspartic proteases secreted by Candida spp. are slightly better inhibitors of M-PMV PR. The best parameters were obtained for inhibitors originally designed for MAV PR (In 13–21). We succeeded in selecting quite potent inhibitors of MPMV PR with IC50 values of approximately 60 nM (In 19 and 20). These data support previous biochemical characterization of M-PMV PR [22] showing that the speciWcity and activity of M-PMV PR is closer to that of MAV than to that of HIV-1. Luminescent detection of the proteolytic activity of HIV-1 PR was also used in an assay developed by Deo et al. [29]. In this method, an HIV-1 protease recognition site was inserted into the Cys/Ser mutant of

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Luminometric method for retroviral protease inhibitors / D. Horáková et al. / Anal. Biochem. 345 (2005) 96–101

Table 1 Inhibition of M-PMV PR by inhibitors of aspartic proteases, secreted aspartic proteases of Candida spp., and proteases of human immunodeWciency virus and myeloblastosis-associated virus

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Inhibitor

PR target

RLU

Percentage of inhibition

Indinavir NelWnavir IvaValValPstAlaPstOH Ritonavir BocValValPstAlaOMe Saquinavir IvaValValStaAlaStaOH (pepstatin) BocValPstAlaPstOMe BocValValPstAlaPstOMe BocValValStaAlaStaOH CH3COValValPstAlaPstOMe C6H5CH2COValValPstAlaPstOMe ProCysValPstAlaMetThrOH AcCysValPstAlaMetProOH GlyProValValPstValSerThrAlaNH2 ProTyrValPstAlaMetThrNH2 ThrPheGlnPstAlaGlnPheThrNH2 ProProTyrValCstGlyLeuTyrProNH2 SerHisMetIleStaValGluLysAspNH2 ThrPheCysAlaPstLeuArgGluAlaNH2 ProThrPheGlnAlaChstLeuArgGluAlaProNH2 Bioluminescence of immobilized conjugate BuVer BuVer with dimethyl sulfoxide

HIV HIV Sap HIV Sap HIV asp Sap Sap Sap Sap Sap MAV MAV MAV MAV MAV MAV MAV MAV MAV

115 116 118 122 126 126 126 132 136 137 138 165 190 199 228 244 256 263 263 281 320 351 20 24

32.8 33.0 33.6 34.8 35.9 35.9 35.9 37.6 38.7 39.0 39.3 47.0 54.1 56.7 65.0 69.5 72.9 74.9 74.9 80.1 91.2 100.0 5.7 6.8

IC50(nM)

267

209

111

82 65 62 56

Note. Ac, acetyl (CH3CO); asp, aspartic; Boc, tert-butoxycarbonyl (CH3)3COCO; Chst, cyclohexylstatin ((3S,4S)-4-amino-3-hydroxy-5-cyclohexylpentanyl acid); Cst, cystatine ((3S,4S)-4-amino-3-hydroxy-5-sulfanylpentanyl acid); HIV, human immunodeWciency virus; Iva, isovaleryl ((CH3)2CHCH2CO); MAV, myeloblastosis-associated virus; Pst, phenylstatine ((3S,4S)-4-amino-3-hydroxy-5-fenylpentanyl acid); Sta, statine ((3S,4S)-4-amino-3-hydroxy-5-methylhexanyl acid); Sap, secreted aspartic protease.

apoaequorin. This protein, which forms a stable complex with the chromophoric unit coelenterazine and molecular oxygen, undergoes a conformational change on the addition of Ca2+, and the oxidation of the chromophore leads to the release of CO2 and emission of light. This assay yielded sensitive detection limits for the protease activity and inhibition with speciWc inhibitors. However, the activity of aequorin is inhibited considerably at temperatures higher than 30 °C, and this temperature sensitivity limits the use of this method. This approach also might not reXect steric hindrance of adjacent domains or the overall fold of the whole target protein on the protease cleavage site. Use of the natural, easily cleavable, two-domain substrate of PRs in the luminometric assay, described in this article, eliminates this problem. The major advantage of our simple luminescence method is the high stability of the substrate and subsequently the stability of the whole kit, which consists of substrate immobilized on multiple-well plates. The other advantages of our method presented here are (i) a high sensitivity, (ii) the possible ability to control sensitivity by changing the parameters of the HRP-catalyzed reaction, and (iii) a good selectivity for inhibitors. The results presented here conWrm that this luminometric method can be used for large-throughput testing of inhibitors of PRs.

Acknowledgments We are grateful to Libune Pavlíbková and Milan Soubek for synthesis of inhibitors. This work was supported by the Grant Agency of the Academy of Sciences (Grant A4055304 research projects) supported by the Czech Ministry of Education (Z 40550506, 1M6837805002, 1M6138896301, and MSM 6046137305).

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